The overall objective of the present study was to determine the genetic architecture of anthracnose fruit rot (AFR) resistance caused by the fungus Colletotrichum acutatum in the University of Florida strawberry (Fragaria × ananassa) breeding germplasm. In 2016–2017, 33 full-sib families resulting from crosses between parents with varying levels of AFR resistance were tested. In 2017–2018, six full-sib families resulting from putative heterozygous resistant parents and homozygous susceptible parents were tested. Additionally, a validation population consisting of 77 advanced selections and ten cultivars was tested in the second season. Inoculation was performed using a mixture of three local isolates of the C. acutatumspecies complex. Phenotypes were scored weekly, and genotyping was performed using the IStraw35 Affymetrix Axiom® SNP array. A pedigree-based QTL analysis was performed using FlexQTL™ software. A major resistance locus, which we name FaRCa1, was detected in both seasons with a peak located at 55–56 cM on LG 6B and explaining at least 50% of the phenotypic variation across trials and seasons. The resistant allele exhibited partial dominance in all trials. The FaRCa1 locus is distinct from the previously discovered Rca2 locus, which mapped to LG 7B. While Rca2 is effective against European isolates from pathogenicity group 2, FaRCa1 appears to confer resistance to isolates of pathogenicity group 1.