Background: Meat products could be sources of enter pathogens. Identification of meat species in different foods could help us in molecular epidemiological studies of pathogens transmitted by meat. Objectives: In this study, we targeted cytochrome b for identification of beef in handmade hamburgers. Patients and Methods: A total of 110 raw handmade hamburgers were collected from different areas of Yazd city, Iran, during spring of 2013. Genomic DNA was extracted using the salting out method. The beef cytochrome b gene was amplified using specific primers. Analysis of the amplicons was done with agarose gel electrophoresis usinga100 base pair (bp) DNA ladder. Results: The results showed that among the 110 handmade hamburger samples, 10 (9.09%) samples did not containany cow meat while 100 samples contained cow meat. Conclusions: We used an appropriate molecular method for controlling raw and processed products. Therefore, this study would be useful for control of correct labeling and protection of consumer’s rights.