Fusarium wilt (FW), caused by the soilborne fungus Fusarium oxysporum f.sp. lactucae (FOL), is an economically important disease of lettuce (Lactuca sativa L.). Four pathogenic races of FOL have been reported, though only race 1 is known to exist in the United States. Recently, California coastal lettuce growers have experienced changes in the severity and incidence of FW. Some race 1-resistant cultivars have exhibited susceptibility, whereas some susceptible cultivars have displayed a reduction in disease severity. In order to determine whether such changes in disease patterns are responses to potentially novel variants, we collected FW symptomatic plant samples from commercial fields in Salinas Valley and Santa Maria, recovered the fungus, and conducted a series of pathogenicity tests in controlled conditions over two years (2022 and 2023) using a standard set of FOL race differentials. Pathogenicity tests revealed two new FOL variants, Fol621s and 916, that elicited novel disease reaction patterns on the standard differentials which have never been reported in the United States or other parts of the world. Isolate 916 incited severe FW on race 1-resistant ‘Costa Rica No. 4’, whereas Fol621s was less virulent on race 1-suceptible ‘Banchu Red Fire’. This study provides valuable information critical for the development of FW management strategies, including broad-spectrum resistance breeding efforts against multiple FOL races and novel variants.