Introduction Flixweed is an annual dicotyledonous winter herb from Cruciferae (Brassicaceae) family which grows in many countries of the world. In Iran it is seen in many of wheat and canola producing regions. Flixweed is used for healing a variety of diseases such as measles and smallpox, cough, asthma, edema and tumor. It also has diuretic, anticancer, antipyretic, antioxidant, anthelmintic, analgesic and tonic activities [2]. In temperate and cold regions of Iran, winter cold is one of the most important environmental stresses which affect growth, development and yield of plants. So determination and improvement of freeze tolerance of winter plants such as Flixweed might promote their cultivation and utilization in cold regions. At present measuring the survival after a recovery period which is followed by a freezing test in controlled conditions is a common method for determining the level of plants freeze tolerance. Calculation of LT50 point or critical temperature based on survival percentage of plant is considered as a quantitative and simple method for evaluating the cold tolerance [1]. But sometimes this criterion is not enough alone and other indices (for example dry weight and Reduced dry matter temperature 50% of plants (RDMT50)) are used to achieve a more accurate estimation of cold tolerance level [3]. In spite of numerous medicinal and industrial properties of Flixweed, there is not a lot of information about freeze tolerance of this plant, so the objective of this study was evaluating the freeze tolerance of Flixweed by survival % and some of growth traits after recovery period. Materials and methods In order to evaluate freeze tolerance in some of Flixweed, a factorial experiment was conducted based on completely randomized design with three replications in faculty of agriculture, Ferdowsi University of Mashhad in autumn of 2009. Experimental factors include five ecotypes of Flixweed (Eghlid, Sabzewar, Hamedan, Torbat-e-Jam and Neyshabour) and 10 freezing temperature levels (from zero to -18 with 2 °C intervals). For this aim, Flixweed seeds were cultivated in pot in autumn and they were grown in natural weather until 5-7 leaf stage. Afterward for applying freezing temperatures, plants were transferred to a thermo gradient freezer. The initial temperature of programmable freezer was 5°C; but gradually decreased in a rate of 2°C.h-1 until reached to desired temperatures. When the freezer temperature reached to -2°C, the plants were treated by the Ice Nucleation Active Bacteria (INAB) to help the formation of ice nuclei in them, also to prevent from super-cooling of samples and to ensure that the mechanism of freeze resistance is tolerance not avoidance. After artificial freezing stress applying (which lasted up to two hours for each freezing temperature); Flixweed seedlings were transferred to greenhouse for recovery. Three weeks after freezing stress, survival percentage (SU %), Lethal temperature for 50% of plants according to the survival% (LT50su), number of leaf and reduced number of leaf temperature 50% (RNLT50), number of node and reduced number of node temperature 50% (RNNT50), dry weight and reduced dry matter temperature 50% (RDMT50) were evaluated. Analysis of variance was performed by MSTAT-C software and correlations between data were carried out by MINITAB 16 program. Mean separation was conducted by least significant difference (LSD) test at 1% probability level. Results and Discussion Results showed that Hamedan ecotype had the highest SU% and Neyshabour ecotype had the lowest SU%. In Eghlid and Neyshabour ecotypes, decline of SU% begun from higher temperarures (-8 °C) compared with other ecotypes (-10 °C). This trend was seen for dry weight and number of leaf too. At -12 °C, number of node in Hamedan ecotype decreased 30% approximately than control, while in Eghlid and Neyshabour ecotypes, this reduction was much higher. In this experiment LT50su ranged between -10.2 to -12.1 °C depending on the ecotypes. Ranking of Flixweed ecotypes according to the LT50su, RNLT50, RNNT50 and RDMT50 indices showed that the Neyshabour and Hamedan ecotypes were the most sensitive and tolerant Flixweed ecotypes to freezing stress respectively. In this survey, there was high and positive correlation between survival percentage and dry weight (r=0.69***), in addition there was negative and significant relationship between SU%, LT50su and RNNT50 (r=-0.67*** and r=-0.82*** respectively). Conclusion According to these results, it seems that temperature threshold for winter injury happening in Flixweed is -12 °C. Despite this for better understanding of cold tolerance of Flixweed, further researches are required under controlled and field conditions.