Ninety Capsicum accessions selected from the USDA Capsicum germplasm collection were screened for their capsaicinoids content using gas hromatography with nitrogen phosphorus detection (GC/NPD). Fresh fruits of Capsicum chinense, C. frutescens, C. baccatum, C. annuum, and C. pubescens were extracted with methanol and analyzed for capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. Mass spectrometry of the fruit crude extracts indicated that the molecular ions at m/z 305, 307, and 293, which correspond to capsaicin, dihydrocapsaicin, and nordihydrocapsaicin, respectively, have a common benzyl cation fragment at m/z 137 that can be used for monitoring capsaicinoids in pepper fruit extracts. Capsaicin and dihydrocapsaicin were the dominant capsaicinoids detected. Capsaicin concentrations were typically greater than dihydrocapsaicin. Concentrations of total capsaicinoids varied from not detectable to 11.2 mg fruit(-1). Statistical analysis revealed that accession PI-441624 (C. chinense) had the highest capsaicin content (2.9 mg g(-1) fresh fruit) and accession PI-497984 (C. frutescens) had the highest dihydrocapsaicin content (2.3 mg g(-1) fresh fruit). Genebank accessions PI-439522 (C. frutescens) and PI-497984 contained the highest concentrations of total capsaicinoids.