Understanding the response of permafrost microbial communities to climate warming is crucial for evaluating ecosystem feedbacks to global change. This study investigated soil bacterial and archaeal communities by Illumina MiSeq sequencing of 16S rRNA gene amplicons across a permafrost thaw gradient at different depths in Alaska with thaw progression for over three decades. Over 4.6 million passing 16S rRNA gene sequences were obtained from a total of 97 samples, corresponding to 61 known classes and 470 genera. Soil depth and the associated soil physical-chemical properties had predominant impacts on the diversity and composition of the microbial communities. Both richness and evenness of the microbial communities decreased with soil depth. Acidobacteria, Verrucomicrobia, Alpha- and Gamma-Proteobacteria dominated the microbial communities in the upper horizon, whereas abundances of Bacteroidetes, Delta-Proteobacteria and Firmicutes increased toward deeper soils. Effects of thaw progression were absent in microbial communities in the near-surface organic soil, likely due to greater temperature variation. Thaw progression decreased the abundances of potential bacterial decomposers of recalcitrant carbon (C) (Spartobacteria) in the lower organic soil, but increased the abundances of those (Actinomycetales, Chitinophaga, etc.) in the mineral soil. Such observations may reflect altered soil C sources in the organic and mineral horizons. Specifically, thaw progression could have increased labile C in the organic soil horizon through stimulated plant growth, but decreased labile C in the mineral soil due to microbial respiration.