Date palm is one of the most important economical crops in the world. Sex determination of date palm in early stage is a prerequisite for breeding and cultivation. The aim of this study is to validate RAPD and ISSR markers for sex identification of date palm genotypes grown under Sudan conditions. DNA was extracted from ten seedlings and five male and female plants using CTAB method. Eight primers, six RAPD and two ISSR primers were examined for their validation in sex determination of date palm genotypes. PCR amplification was performed using these primers. Four RAPD primers OPA02, OPJ-09, RD A02 and RD A21 were amplified male specific band with size of 1000, 1100, 1000 and 1400 pb respectively, while ISSR markers could not. The specific bands were observed clearly among all male genotypes and absent in female samples unknown samples irrespective of genotypes. Our results could be useful for sex determination of date palm sex in seedling stage and would promote date palm cultivation and production.