Granulomatous/pyogranulomatous nodules that are not neoplastic and for which no organism can be cultured or identified by histological examination are a diagnostic and therapeutic challenge. Sterile pyogranuloma/ granuloma syndrome (SPGS) is such a disorder, but it is possible that organisms (eg, Leishmania spp, Mycobacterium spp, Nocardia spp, Actinomyces spp, Serratia spp) may provide persistent antigenic stimulation in some cases and cause a granulomatous inflammatory reaction. In this study, 40 canine skin biopsies diagnosed as sterile lesions with a granulomatous/pyogranulomatous pattern were reevaluated; real-time PCR was used to detect Leishmania spp, Mycobacterium spp, Nocardia spp, and Serratia marcescens. In addition, special stains and anti-Bacillus Calmette–Guerin (BCG) and Leishmania spp immunohistochemistry were performed. Histological review, special stains, and immunohistochemistry did not reveal any infectious agents. Real-time PCR was negative for Mycobacterium spp and Nocardia spp but positive for Leishmania spp in 4 biopsies and S marcescens in 2 samples. The cases with positive results were not histologically distinct from those with negative findings. These results provide further evidence that diagnosis of noninfectious granulomatous/ pyogranulomatous skin lesions could result from chronic antigenic stimulation from pathogens in skin lesions.