Fusarium wilt of lettuce is caused by the pathogen Fusarium oxysporum f. sp. lactucae (Fol) and is a growing threat to global lettuce production. Fol was first detected in Florida in 2017 and was subsequently confirmed as race 1. Management strategies for this long-persisting soil pathogen are limited, time-consuming and expensive, and they may lack efficacy. Identifying diverse sources of genetic resistance is imperative for breeding adapted cultivars with durable resistance. The objectives of this study were to identify sources of resistance against a race 1 isolate of Fol in Florida, delineate the relationship between foliar and taproot symptoms, and investigate the inheritance of resistance and partial resistance in two F2 populations. Thirteen experiments were conducted in greenhouse and field locations to characterize the diversity of genetic resistance in the genus Lactuca. Leaf cultivars Dark Lollo Rossa and Galactic; romaine breeding lines 43007, 60182, and C1145; and iceberg breeding line 47083 consistently exhibited low foliar and taproot disease symptoms. Resistance was not identified among the wildtype Lactuca or primitive plant introductions (PI) in this study based on taproot symptoms. An additional test was conducted to study the segregation pattern of Fol resistance between one resistant and one susceptible accession (R × S) and one partial resistant and one susceptible accession (PR × S). The F2 population from ‘60182 × PI 358001-1’ fit the expected segregation ratio for a single recessive locus model, whereas the ratio for ‘Dark Lollo Rossa × PI 358001-1’ did not fit either recessive or dominant single locus models. These sources of resistance are potential candidates for developing commercial cultivars with multiple resistance loci against Fol race 1, especially for the Florida lettuce production system.