Carbon dioxide fixation is one of the most important biogeochemical processes worldwide, but our current understanding of the distribution of microbial autotrophy and its ecological significance in oligotrophic freshwater systems, and particularly in benthic habitats, is poor and mainly limited to photoautotrophic organisms. In this study, we investigated the autotrophic microbial communities inhabiting the sediments of a high elevation, oligotrophic freshwater pond in the North-Western Italian Alps. The abundance and distribution of three different forms of the RubisCO large-subunit gene were assessed in samples collected at different depths by qPCR, and correlations with sediment geochemical properties and total bacterial abundance were also examined. RubisCO forms cbbLG, cbbLR and cbbM were all detected, with abundances of 9.13-10.90, 6.93-8.77 and 6.75-7.93 Log copies per g of dry weight, respectively. For all of them interannual variability overcame depth-related variability. RubisCO genes abundance was strongly correlated with total bacterial abundance, and both of them were positively correlated with Ca2 + and Mg2 + concentration. These observations provide some first indications on the distribution of photo-and chemolithoautotrophic bacteria relying on the Calvin-Benson-Bassham (CBB) cycle for C fixation in alpine pond sediments, and suggest that they may represent an important component of the total benthic microbial community.