It is important to identify livestock breeds for breeding programs. The identification issue of livestock can be resolved by using molecular identification tools that acceptable to preserve and maintain pure breeds worldwide. The application of molecular identification methodology is more important for developing nations such as Pakistan, where uncontrolled crossbreeding has become a common practice and import of exotic animals and germplasm is ever increasing. This is always keeping local breeds at risk that is also an FAO concern. Therefore, the current study was designed to develop standard molecular markers for Cholistani cattle to ascertain their purity for breeding purpose. In this study, 50 and 48 unrelated males were sampled for each Cholistani and Crossbred cattle respectively. Candidate molecular markers present in Cholistani but absent in Crossbred and vice versa were detected using amplified fragment length polymorphism method. Eleven markers were developed and were converted to single nucleotide polymorphism markers for high throughput genotyping. The allele frequencies in both breeds were determined for discrimination ability using PCR-AFLP. The probability of identifying Cholistani breed was 0.981 and probability of misjudgment was 0.313 using single selected markers. However, probability for judgment and misjudgment with 2 markers and combined with 3 markers was 0.740 and 0.362 and 0.995 and 0.562, respectively. The identified markers can ascertain breed identification and facilitate the breed improvement program through genetic selection in the country.