India has 34 recognized breeds of cattle in addition to many more not characterized and accredited so far. It is imperative to characterize all the cattle germplasm of the country so as to have better breeding and conservation options. Thus, present study was planned for assessing genetic diversity and relationship between three local cattle populations (Gangatiri, Shahabadi and Purnea) and two established cattle breeds (Bachaur and Siri) of eastern India by using 21 FAO and ISAG recommended microsatellite markers. A total of 243 unrelated DNA samples of five cattle populations were collected from respective habitats. A total of 304 microsatellite alleles were identified with number of alleles at one locus ranging from 5 to 29. The average observed heterozygosity lie within the narrow range of 0.681 ± 0.04 in Purnea to 0.721 ± 0.03 in Siri. Mean estimates of observed and expected heterozygosity over all loci and breeds were 0.704 ± 0.02 and 0.720 ± 0.01, respectively. In the overall population, the homozygote excess (FIT) of 0.073 ± 0.02, was partly due to the homozygote excess within breeds (FIS = 0.026 ± 0.02) and to a larger extent due to genetic differentiation among breeds (FST = 0.048 ± 0.01). The genetic distance, STRUCTURE and Principal Component Analyses concluded that the Siri cattle are most distinct among the investigated cattle populations. Furthermore the analysis of genetic structure indicated that the most probable number of clusters is four. All analysis showed that a significant amount of genetic variation is maintained in local cattle populations of which Shahabadi and Purnea are distinct from the recognized breeds of the area and needs recognition as breeds.