High elevation, mountainous regions have a high concentration of mining activities and resulting acid mine drainage (AMD) that is typically acidic and often contains elevated concentrations of metals. The impacts of AMD on denitrifying microbial communities is not well understood, despite these organisms’ central role in the nitrogen cycle, contribution to greenhouse gas production, and potential to provide ecosystem services through the mitigation of nitrogen pollution. This study examined denitrifying microbes across four regions within the Iron Springs Mining District (13 sites over four time-points) located in Southwest Colorado at high elevation that receive AMD or naturally-occurring acid rock drainage (ARD). Denitrification functional gene sequences ( nirS and nirK coding for nitrite reductase) had a high number of observed OTUs (260 for nirS and 253 for nirK) and were observed at sites with pH as low as 3.2, dissolved oxygen as low as 1.0 mg/L, and metals >10 mg/L (including aluminum, iron, manganese, and zinc). A majority of the nirK and nirS OTUs (> 60%) were present in only one sampling region. Approximately 8% of the nirK and nirS OTUs had a more cosmopolitan distribution with presence in three or more regions. Phylogenetically related OTUs were found across sites with very different chemistry. The total nirS community structure was correlated to iron, conductivity, sodium, and calcium, which may suggest that these factors play an important role in shaping the nirS community. Overall, these findings improve upon our understanding of the potential for denitrification within an ecosystem impacted by AMD and provide a foundation for future research to understand the rates and physiology of these denitrifying organisms.