The aim of the present study is to determine the effect of antiepileptic drug (Phenytoin) on the mitochondrial CYT b gene in mice fetus. The used mice were arranged in four different groups: (G1) control group, (G2) treated males with untreated females, (G3) treated females with untreated males and (G4) treated males with treated females. All treated groups were interperitoneally injected with a dose of phenytoin 8mg/kg for 15 days before mating .The injection continuously with pregnant females at 21th day of gestation,. At delivery the fetuses are collected .In this study, the molecular techniques investigate the effect of the drug (in each group) on CYT b gene of mice fetus at delivery by PCR, RFLPs and PCR-RFLP techniques. The restriction fragments length polymorphisms (RFLP) enabled scientific identification and genetic differentiation between the studied groups. The polymerase chain reaction (PCR) facilitates amplification and analysis of fragments or DNA genes. The selected CYT b gene of each group has been amplified by using PCR technique. RFLP profiles of genes were obtained by digestion with twelve restriction enzymes (ApoI,BseRI,PstI , AfeI, SpeI, EcoRI, DraI, AseI, BanI, HindIII , AcsI and MaeIII). This approach has enabled us to predict the mutation that occurs on "CYT b"gene of the mice embryo of the different studied groups.