AbstractThe aim of this research was to assess the SRY-gene polymorphism in Madura cattle populations and the hybrids of Madura cow with Limousin bull. We investigated the specific markers of the paternally transmitted Y-chromosome for the direct detection of male-mediated introgression. We performed polymerase chain reaction and restriction fragment length polymorphisme (PCR– RFLP) to identify the polymorphism of Y-chromosomes in 17 Madura cattle from Pamekasan district, 4 conserved Madura cattle from Sapudi Island and 22 Limousin x Madura crossbred cattle from Pamekasan. A 211bp PCR product were cut with PstI and BfaI restriction enzyme. The result showed all the Madura cattle types were identified by PstI resrtiction enzyme performed into two fragments DNA. The polymorphisme of Y-chromosome was recognized by BfaI restriction enzymes at the position 432bp and 544bp. Using BfaI restriction enzyme, this study identified 68.18% of Limousin x Madura crossbred cattle was in the sequence of Y-chromosome. The high numbers of BfaI site restriction in the sequence of Y-chromosome due to the introduction Limousin bull by artificial insemination. For those, the preservation of pure Madura cattle as one of animal genetics resources in Indonesia should be a concern.