The anther culture response of Citrullus lanatus L. was studied to evaluate the effect of growth regulators and wheat ovary-conditioned medium in Charleston Gray cultivar and the effect of temperature pretreatment in Crimson sweet cultivar. Callus-mediated embryogenesis was induced on Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid (2,4-D) and a-naphthaleneacetic acid alone as well as 2,4-D in combination with N6-benzylaminopurine/kinetin (BAP/KN). Optimal percentage of embryogenic calli (EC) and mean number of embryos per anther (ME/A) were induced on MS medium supplemented with 2.0 µM 2, 4-D and 1.5 µM BAP in cultivar of Charleston Gray. In this cultivar, the inclusion of 10 wheat ovaries in the induction medium significantly increased the percentage of EC and ME/A up to 2.5- and 3-fold when compared with that in the control, respectively. Low-temperature pretreatment of flower buds (4 °C for 2 and 5 days) and cultured anthers (4 °C for 4 and 8 days) were tested in Crimson sweet cultivar and best response in terms of %EC and ME/A was obtained with pretreatment of cultured anthers at 4 °C for 4 days and flower buds at 4 °C for 2 days. Also, heat pretreatments of the cultured anthers at 30 °C for 7 and 32 °C for 2 days produced the highest percentage of EC compared to control and other heat pretreatments in Crimson sweet cultivar. The highest frequency of ME/A was obtained from cultured anthers incubated at 32 °C for 2 days. Embryos germinated into plantlets on MS medium containing 2.22 µM BAP, 3 % sucrose, and 0.8 % agar. The root tips of 12 regenerated plantlets were analyzed for ploidy level, of which 10 have been haploids.