Chemosynthetic primary production supports hydrothermal vent ecosystems, but the extent of that productivity and its governing factors have not been well constrained. To better understand anaerobic primary production within massive vent deposits, we conducted a series of incubations at 4, 25, 50 and 90 °C using aggregates recovered from hydrothermal vent structures. We documented in situ geochemistry, measured autochthonous organic carbon stable isotope ratios and assessed microbial community composition and functional gene abundances in three hydrothermal vent chimney structures from Middle Valley on the Juan de Fuca Ridge. Carbon fixation rates were greatest at lower temperatures and were comparable among chimneys. Stable isotope ratios of autochthonous organic carbon were consistent with the Calvin–Benson–Bassham cycle being the predominant mode of carbon fixation for all three chimneys. Chimneys exhibited marked differences in vent fluid geochemistry and microbial community composition, with structures being differentially dominated by gamma (γ) or epsilon (ε) proteobacteria. Similarly, qPCR analyses of functional genes representing different carbon fixation pathways showed striking differences in gene abundance among chimney structures. Carbon fixation rates showed no obvious correlation with observed in situ vent fluid geochemistry, community composition or functional gene abundance. Together, these data reveal that (i) net anaerobic carbon fixation rates among these chimneys are elevated at lower temperatures, (ii) clear differences in community composition and gene abundance exist among chimney structures, and (iii) tremendous spatial heterogeneity within these environments likely confounds efforts to relate the observed rates to in situ microbial and geochemical factors. We also posit that microbes typically thought to be mesophiles are likely active and growing at cooler temperatures, and that their activity at these temperatures comprises the majority of endolithic anaerobic primary production in hydrothermal vent chimneys.