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The Madura cattle is one of Indonesian that have the advantages. One of the genes regulating genital formation in males of Madura cattle is the Sex-determining Region Y gene (SRY), this gene is dominant in regulating sex determination and fertile markers in male cattle. This study aims to determine the genetic variation of the SRY gene DNA sequences in local male Madurese cattle and analyze the function of these genes. The research method used for total blood DNA isolation was the phenol chloroform method. The SRY gene amplification samples were DNA sequenced and analyzed bioinformatics. The results of the SRY PCR gene electrophoregram show bands with a length of + 600pb. Five different regions on Madura Island were used as sampling locations, namely Lombang, Pajudan, Pasean, Kanang and Kanwa. The occurrence of transversion mutations in the 4th base sequence with the monomorphic pattern and prediction of the polypeptide showed the same polypeptide results from the two samples, namely AP-1 Complex. This protein has an important role as a transcription factor in the normal development of the testis. The sequence variation obtained is able to bring about the nature of heterozygosity and increase the value of genetic diversity.