Verticillium dahliae is a pathogen of spinach (Spinacia oleracea) during spinach seed crop production but not in vegetative leafy spinach crops, because plants remain asymptomatic until bolting has been initiated (conversion from vegetative to reproductive growth). The objectives of this research were to evaluate a set of USDA spinach germplasm accessions for resistance to Verticillium wilt and to determine the range in incidence of natural seed infection/infestation in a subset of the USDA spinach accessions screened for resistance. A total of 120 Spinacia spp. accessions from the USDA North Central Regional Plant Introduction Station spinach germplasm collection and 10 commercial S. oleracea hybrids were screened for resistance to V. dahliae in Trials 1, 2, and 3 in 2006, 2007, and 2008, respectively, in greenhouse evaluations. Each accession was grown in either V. dahliae-infested or non-infested potting mix and rated weekly on a seven-step scale from 1 week before bolting to 4 weeks after bolting. A wide range of disease severity ratings was observed among the accessions evaluated. Most of the accessions were highly susceptible. There was no evidence of qualitative resistance but some showed greater levels of quantitative resistance than others. Plants in soil infested with V. dahliae senesced faster and had significantly reduced biomass compared with plants in non-infested soil of the same accession. In addition, in Trial 2 (2007), 34% (20 of 59) of the seed samples assayed of the accessions were infested or infected with V. dahliae; and in Trial 3, (2008) 16% (21 of 130) of the seed samples of the USDA accessions evaluated were infested or infected with V. dahliae, V. tricorpus, or Gibellulopsis nigrescens (formerly known as V. nigrescens). These results are valuable for characterizing potential genetic variability within spinach germplasm for resistance to V. dahliae.