Breeds can be easily identified using molecular tools based on genomic architecture of breed within a species. Moreover, in developing countries like Pakistan where the danger of genetic erosion is a serious threat due to uncontrolled crossbreeding, the application of such tools is promising for breed identification and conservation. Thus, the current research was planned to prepare AFLP based genetic markers for distinction of Red Sindhi cattle from crossbred population. In current study, 50 and 48 unrelated (males as well as females) were sampled for Red Sindhi and crossbred cattle populations, respectively. AFLP method was used to fetch prospective molecular markers present only in Red Sindhi however, lacking in crossbred and vice versa. The analysis generated eleven markers that were translated to single nucleotide polymorphism markers for genotyping. These markers were used for allelic variation analysis in both sampled populations. Moreover, single selected markers generated an identification probability of 0.800 while a probability for misjudgment of 0.313 for Red Sindhi cattle. However, the combination of three markers yielded a probability for judgment of 0.941 while a probability of misjudgment was 0.413. The study yielded panel of AFLP markers for identification of Red Sindhi that can be used as breed purity test to discriminate between Red Sindhi and crossbred population.