The structure and function of microbial communities inhabiting the subseafloor near hydrothermal systems are influenced by fluid geochemistry, geologic setting, and fluid flux between vent sites, as well as biological interactions. Here we used genome‐resolved metagenomics and metatranscriptomics to examine patterns of gene abundance and expression and assess potential niche differentiation in microbial communities in venting fluids from hydrothermal vent sites at the Mid‐Cayman Rise. We observed similar patterns in gene and transcript abundance between two geochemically distinct vent fields at the community level, but found that each vent site harbors a distinct microbial community with differing transcript abundances for individual microbial populations. Through an analysis of metabolic pathways in 64 metagenome‐assembled genomes (MAGs), we show that MAG transcript abundance can be tied to differences in metabolic pathways and to potential metabolic interactions between microbial populations, allowing for niche‐partitioning and divergence in both population distribution and activity. Our results illustrate that most microbial populations have a restricted distribution within the seafloor, and that the activity of those microbial populations is tied to both genome content and abiotic factors.