Downy brome (Bromus tectorum L., syn. cheatgrass) is a winter annual grass that invades North American cropping, forage, and rangeland systems. Control is often difficult to achieve, because B. tectorum has a large seedbank, which results in continuous propagule pressure. Pyrenophora semeniperda (Brittlebank and Adam) Shoemaker, a soilborne fungal pathogen, has been investigated as a biological control for B. tectorum, because it can kill seeds that remain in the seedbank, thereby reducing propagule pressure. Temperature influences P. semeniperda and has not been investigated in the context of seeds collected from different B. tectorum locations, that may vary in susceptibility to infection. We compared the effects of temperature (13, 17, 21, 25 C) and B. tectorum seed locations (range, crop, subalpine) with different mean seed weights on infection rates of P. semeniperda using a temperature-gradient table. Infection differed by seed location (P < 0.001) and temperature (P < 0.001), with lighter-weight seeds (i.e., range and subalpine) more susceptible to P. semeniperda infection. Infection increased as temperature increased and was higher at 21 C (66.7 ± 6.7%) and 25 C (73.3 ± 6.0%). Germination was affected by seed location (P < 0.001) and temperature (P = 0.019). Germination was highest for the crop seed location (45.4 ± 4.2%) and overall decreased at higher temperatures (21 and 25 C). Our results suggest that B. tectorum seeds from a crop location are less affected by P. semeniperda than those from range and subalpine locations. Moreover, this demonstrates a temperature-dependent effect on all populations.