The present study aims to understand the existing genetic diversity and structure of six native cattle breeds Rathi, Tharparkar, Nagori, Mewati, Gir, and Kankrej) adapted to the north-western arid and semi-arid region of India based on microsatellite loci. Various diversity estimates, mean number of alleles 12.84); effective number of alleles 5.02); gene diversity 0.769), and observed heterozygosity 0.667) reflected the existence of substantial within-breed diversity in all the investigated cattle breeds. Mean estimates of F-statistics: F(IT)=0.144 +/- 0.023, F(IS)=0.071 +/- 0.021, and F(ST)=0.078 +/- 0.014 were significantly different from zero P<0.05). The interbreed relationships indicated moderate level of breed differentiation between the six cattle breeds with least differentiation between Kankrej-Mewati pair. The phylogeny structuring further supported close grouping of Kankrej and Mewati breeds. Correspondence analysis plotted Rathi, Tharparkar, and Gir individuals into three separate areas of multivariate space; whereas, Kankrej, Mewati, and Nagori cattle showed low breed specific clustering. This reflected the existence of discrete genetic structure for Tharparkar, Rathi, and Gir, the prominent dairy breeds of the region; whereas, admixture was observed for Kankrej, Mewati, and Nagori individuals.