Abstract Methanotrophic bacteria in soil serve as the only known biological sink of atmospheric methane, and impact net methane emission from soils. Most members of methanotrophs use particulate methane monooxygenase enzyme, encoded by the pmoA gene, to oxidize methane. Here, Illumina sequencing (iTAG) of pmoA gene was carried out to examine methanotrophic bacterial diversity in soils under different land use. Additionally, the effect of soil-type on pmoA amplicon sequence abundance was analyzed. Two sites in Ohio, each comprising of long-term no-tillage plots, plow-tillage plots, grassland, and forest were sampled. The tillage plots also included continuous-corn and corn-soybean crop rotations. By referencing a curated database of the pmoA gene, we were able to identify eight genera of methanotrophs with varying relative abundances in the samples. Additionally, four genera were differentially abundant across samples (alpha = 0.005) and included Methylocystis , Methylocococcus , Methylosoma , and USCa . About 30% of processed sequences were unclassified. Nonmetric multidimensional scaling (NMDS) of unweighted UniFrac and Bray-Curtis metrics showed clear community distinction based on sites. Soil type had the most significant effect (p