The present study was designed to assess the presence/absence and frequency of Barr Bodies on Polymorph Nuclear Neutrophils (PMNs) as a confirmatory guide towards sex determination in cattle and buffalo. Blood samples of adult cattle (n=26) and buffaloes (n=26) grouped as males and females (n=13 each), were collected from the jugular vein through appropriate measures. Thin blood smears were stained using Field stain „A‟ and „B‟. The PMNs were counted and observed for Barr Bodies attached to them. Only the neutrophils with terminable lobes were examined and counted. The mean value ±SE of the Barr Bodies was calculated and the difference between males and females of both species was analyzed through independent T-test. The mean ±SE values for Barr Bodies were significantly higher (P≤0.05) in female cattle (9.61±1.11) as compared to their male counterparts (0.92±0.28). Similar results were revealed for buffaloes being 6.23±1.15 and 0.92±0.28 for females and males, respectively. In a nutshell, it is concluded that female cattle and buffaloes have a higher occurrence of Barr Bodies on their PMNs as compared to males. Furthermore, it can also be deduced that sex determination in cattle and buffalo through this cyto-diagnostic technique is quiet certain and precise.