The McMurdo Dry Valleys (MDV) in Antarctica harbor a diverse assemblage of mat-forming diazotrophic cyanobacteria that play a key role in nitrogen cycling. Prior research showed that heterotrophic diazotrophs also make a substantial contribution to nitrogen fixation in MDV. The goals of this study were to survey autotrophic and heterotrophic diazotrophs across the MDV to investigate factors that regulate the distribution and relative ecological roles of each group. Results indicated that diazotrophs were present only in samples with mats, suggesting a metabolic coupling between autotrophic and heterotrophic diazotrophs. Analysis of 16S rRNA and nifH gene sequences also showed that diazotrophs were significantly correlated to the broader bacterial community, while co-occurrence network analysis revealed potential interspecific interactions. Consistent with previous studies, heterotrophic diazotrophs in MDV were diverse, but largely limited to lakes and their outlet streams, or other environments protected from desiccation. Despite the limited distribution, heterotrophic diazotrophs may make a substantial contribution to the nitrogen budget of MDV due to larger surface area and longer residence times of lakes. This work contributes to our understanding of key drivers of bacterial.