The last decade has witnessed growth in scientific interest towards studying the biodiversity and ecology in extreme environments. Acid Mine Drainage (AMD), formed through the bio-oxidation of metal sulphides, is a typical extreme environment that is inhospitable to most life forms. Through the application of traditional culture-based molecular techniques, it has been established that AMD is home to a low diversity, specialized and novel microbial community that plays a critical role in its formation. Intensive efforts have been channeled towards understanding the ecology, microbial biodiversity, processes and metabolic networks within these simple ecosystems. Molecular techniques using high-throughput next-generation sequencing platforms have taken center stage in discerning both the taxonomic and functional diversity in these microbial communities. Recently developed post-genomic approaches as well have been particularly instrumental in deciphering in situ interactions within microbial communities and the environment. In this review, a critical synopsis of the current applications of advanced molecular techniques in probing microbial and functional diversity in AMD environments is presented.