Brucella could recovered in 10 samples of vaginal swabs, abortion and placenta (two from cattle, one from buffaloes, four from goats, and one from a bitch) of the 248 samples processed by cultural, morphological, biochemical characteristics. Among isolates, eight from abortion cases (two from cow, one from buffalo, four from goat and one from a bitch) and two isolates from reproductive disorder (one from a cow and one from a buffalo) were recovered. Three different sets of primers were compared for the detection of Brucella species. The three pairs of primers amplified three different fragments viz., (i) B4/B5 primer pair amplified a 223 bp (ii) F4/R2 primer pairs amplified a 905 bp (iii) JPF/JPR primer pair amplified a 193 bp. Of these primer pairs, B4/B5 was found to be more sensitive as it detected 10 Brucella isolates. Whereas the other two primer pairs, F4/R2 and JPF/JPR, detected eight samples of Brucella organisms. The isolates identified as Brucella organisms were subjected to species differentiation using combinatorial PCR to identify the species of genus Brucella simultaneously. Four pairs of primers targeting the gene encoding cell surface protein (BCSP31) and outer membrane protein (omp2b, omp2a and omp31) were used. PCR using these primers gaves rise to a specific pattern of amplification for each Brucella species. Out of 10 isolates of Brucella,the five isolates from cattle and buffaloes could be identified as B. abortus when fragments of BCSP31 and omp2b/2a were amplified by B. abortus-specific primers, whereas isolates from goats could be identified as B. melitensis by the amplification of fragments of BCSP31, omp2b/2a and omp31 using primer B4/B5, JPF/JPR-ab and omp31. Identification of B. canis from the bitch isolates could be made by amplification of BCSP31 and omp31.