A total of thirty-eight Mafriwal cattle were selected from a localcattle herd of a government cattle farm; of which 36 animals were sub-fertile Mafriwal female dams and two bulls which were considered as control animals (one male Mafriwal and one male Jersey). Two markers were used in the detection of Y chromosome in the sub-fertile female animal which are testis specific proteins Y-encoded (TSPY) and amelogenin (AMLX/AMLY) genes. The genes were amplified using PCR. The DNA bands from a normal male for TSPY gene size was approximately 260 bp while AMLX/ AMLY gene were approximately 341 and 467 bp. The examination of all samples showed that the sub-fertile cow revealedonly 467 bp while three fragments were detected in the control group; 260 bp (testis specific protein, Y-encoded gene), 341 and 467 bp (Amelogenin gene). The results showed that the sex chromosomeanomalies associated with Y chromosome did not occur in this group. These two sex markers can be used for the diagnosis of Y chromosome abnormality in a sub-fertile cow through polymerase chain reactionwhich is a rapid and reliable method for use in breeding herds.