Abstract Temperature sensitivity of community-level physiological profiles (CLPPs) was examined for two semiarid soils from the southwestern United States using five different C-substrate profile microtiter plates (Biolog GN2, GP2, ECO, SFN2, and SFP2) incubated at five different temperature regimes. The CLPPs produced from all plate types were relatively unaffected by these contrasting incubation temperature regimes. Our results demonstrate the ability to detect CLPP differences between similar soils with differing physiological parameters, and these differences are relatively insensitive to incubation temperature. Our study also highlights the importance of using both bacterial and fungal plate types when investigating microbial community differences by CLPP. Nevertheless, it is unclear whether or not the differences in CLPPs generated using these plates reflect actual functional differences in the microbial communities from these soils in situ.