The objective of this study was to compare the efficacy of eight Blastomyces dermatitidis yeast phase lysate antigens (T-58: dog, Tennessee; T-27: polar bear, Tennessee; ERC-2: dog, Wisconsin; B5894: human, Minnesota; SOIL: soil, Canada; B5896: human, Minnesota; 48089: human, Zaire; 48938: bat, India) in the detection of the immunoglobulins IgG and IgM in serum specimens from canines with blastomycosis. An indirect enzyme-linked immunosorbent assay (ELISA, peroxidase system) was used to analyze sera collected during four different intervals post-infection. The yeast lysate antigen 48938 was a reactive antigen for the detection of both IgG (mean absorbance value range: 1.198–2.934) and IgM (mean absorbance value range: 0.505–0.845). For the same sera, antigen T-27 was also effective in the detection of IgG (mean absorbance value range: 0.904–3.356) and antigen 48089 was useful for the detection of IgM (mean absorbance value range: 0.377–0.554). The yeast lysate antigen B5894 proved to be a poor antigen for the detection of both IgG and IgM (mean absorbance value ranges: 0.310–0.744 for IgG, 0.025–0.069 for IgM). Inherent variations in yeast lysate antigens such as these may be utilized to develop improved immunoassay procedures for the specific detection of IgG or IgM in cases of blastomycosis.