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Genetic diversity among microbial populations in six different age series coal mine overburden spoil and nearby forest soil was evaluated through culture-independent approach using RAPD markers– a PCRbased DNA fingerprinting technique. Twenty primers were used for amplification of metagenomic DNA, and banding patterns of 21 genotypes were compared to assess microbial community structure among seven different soil profiles. The Nei’s genetic diversity and Shannon’s information index was estimated to be 0.3558 and 0.5206 respectively. Besides, the population diversity and estimated gene flow were observed to be 0.1181 and 0.2482 respectively. Dendrogram analysis based on neighbor joining method and bootstrapping procedure revealed that 21 genotypes were well segregated into three independent clusters. Analysis of molecular variance (AMOVA) indicated 55.2% genetic variability among population was attributed to different soil profiles, where as 44.8% variability was accounted within population attributed to different habitats. The study correlated well with temporal and spatial variations among different age series coal mine overburden spoil and nearby forest soil confirming the PCR-based DNA fingerprinting technique using RAPD markers are useful means of discriminating microbial communities and estimating relatedness. Further, the analysis revealed gradual enrichment in the existing genetic composition due to reclamation over time supporting greater microbial diversity.