Fusarium wilt of tomato (Solanum lycopersicum), caused by fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), is one of the most important diseases in tomato production. Three races of the pathogen are described, and race-specific resistance genes have been applied in commercial tomato cultivars for controlling the disease. Race 3 (Fol3) threatens tomato production in many regions around the world, and novel resistance resources could expand the diversity and durability of Fol resistance. The wild tomato species, Solanum pennellii, is reported to harbor broad resistance to Fol and was the source of two known Fol3 resistance genes. In this study, we evaluated 42 S. pennellii accessions for resistance to each fusarium wilt race. F1 plants, developed from crossing each accession with the Fol3 susceptible line ‘Suncoast’, were evaluated for Fol3 resistance, and BC1F1 plants were screened to determine the likelihood that Fol3 resistance was based on a novel locus (loci). Nearly all accessions showed resistance to Fol3, and many accessions were resistant to all races. Evaluation of F1 plants indicated a dominant resistance effect to Fol3 from most accessions. Genetic analysis indicated 24 accessions are expected to contain one or more novel Fol3 resistance loci other than an allele near the I-3 locus. To investigate genetic structure of the S. pennellii accessions used in this study, we genotyped all 42 accessions using genotyping by sequencing. Approximately 20% of the single nucleotide polymorphism (SNP) loci were heterozygous across accessions, likely due to the outcrossing nature of the species. Genetic structure analysis at 49,120 unique SNP loci across accessions identified small but obvious genetic differentiations.