The present study deals with genetic diversity analysis of Labeo rohita collected from hatchery stock and Nanak Sagar reservoir of Uttarakhand through microsatellite marker. Total 20 microsatellite primers were designed by using software Primer-BLAST and Primer-3. A total of 12 microsatellite loci were successfully amplified. After performing native PAGE using amplified 50 DNA samples each, POP GENE Version 1.32 was used to calculate microsatellite variation. The average expected Nei's genetic diversity ranged from 0.328 to 0.529 with mean value of 0.458 for Labeo rohita across all loci from hatchery whereas the average expected gene diversity ranged from 0.510 to 0.749 with mean value of 0.630 for Labeo rohita across all loci from Nanak Sagar reservoir. The observed and expected heterozygosity ranged from 0.2237 to 0.3326 and 0.2786 to 0.3763 respectively for Labeo rohita from hatchery. The mean value of observed heterozygosity was 0.2864 and that of expected heterozygosity was 0.3238. Fis values were found to be positive at all loci in hatchery with mean value of 0.256. The Fis values were found to be negative at all loci in Nanak Sagar with mean value of 0.176. The observed and expected heterozygosity ranged from 0.4469 to 0.5459 and 0.4829 to 0.5561 respectively for Labeo rohita from Nanak Sagar with mean value of 0.4894 and 0.5151 for observed and expected heterozygosity. Mean values for Shannon's information index for all microsatellite loci were 1.1091 for hatchery and 1.2062 for Nanak Sagar reservoir population. Slightly more level of observed heterozygosity in Labeo rohita from reservoir than Labeo rohita from hatchery might be due to presence of more differentiated stocks in reservoir. Lesser value of observed heterozygosity in Labeo rohita from hatchery than reservoir might be possibly due increase in incidents of inbreeding in successive generations owing to lack of regular germplasm exchange of appropriate genetic diversity. The microsatellite analysis showed that Labeo rohita of Nanak Sagar reservoir is more genetically diversed and genetically differentiated as compared to Labeo rohita from hatchery.