Tropical peatlands play a critical role in global carbon storage and greenhouse gas flux, yet the role of microbial communities in these ecosystems remains poorly understood. Methane-oxidizing bacteria (MOB) are considered an efficient biological filter for methane and can mitigate its release into the atmosphere, facilitating an ecosystem’s capacity to become a net sink. Prokaryotic gene amplicon surveys targeting a unique biomarker instead of a universal one (i.e., 16S rRNA) can reveal a more comprehensive analysis of microbial communities with ecological functions (i.e., methanotrophy). The alpha subunit of particulate methane monooxygenase (pmoA) is commonly targeted as a phylogenetic biomarker for both aerobic and anaerobic MOB. Here, we tested three different primer sets and investigated their ability to assess methanotrophic diversity across three biogeochemically distinct tropical peatland sites in the Pastaza-Marañón foreland basin (PMFB) in western Amazonia. The results showed that sequencing using 16S rRNA and pmoA genes revealed differences in MOB taxonomic identification in 21 tropical peat soils. Beta diversity analysis of pmoA genes suggests that site location is not the main driver of differences in MOB community makeup. This work offers insight into the strengths and weaknesses of targeted gene amplicon surveys using 16S and pmoA from tropical peat soils as a case study.