© 2017 The American Phytopathological Society. Fusarium oxysporum species complex (FOSC) isolates were obtained from celery with symptoms of Fusarium yellows between 1993 and 2013 primarily in California. Virulence tests and a two-gene dataset from 174 isolates indicated that virulent isolates collected before 2013 were a highly clonal population of F. oxysporum f. Sp. Apii race 2. In 2013, new highly virulent clonal isolates, designated race 4, were discovered in production fields in Camarillo, California. Long-read Illumina data were used to analyze 16 isolates: six race 2, one of each from races 1, 3, and 4, and seven genetically diverse FOSC that were isolated from symptomatic celery but are nonpathogenic on this host. Analyses of a 10-gene dataset comprising 38 kb indicated that F. oxysporum f. Sp. Apii is polyphyletic; race 2 is nested within clade 3, whereas the evolutionary origins of races 1, 3, and 4 are within clade 2. Based on 6,898 single nucleotide polymorphisms from the core FOSC genome, race 3 and the new highly virulent race 4 are highly similar with Nei's Da = 0.0019, suggesting that F. oxysporum f. Sp. Apii race 4 evolved from race 3. Next generation sequences were used to develop PCR primers that allow rapid diagnosis of races 2 and 4 in planta.