We determined the prevalence of natural field infections of the fire ant pathogen Thelohania solenopsae Knell, Allen, and Hazard in the monogyne and polygyne social forms of the red imported fire ant, Solenopsis invicta Buren, in three pastures in Florida. Social form was determined by examining the genotype of ants at the Gp-9 locus. The monogyne form contains a single fertilized queen per colony, and colony members have a genotype of Gp-9BB. In contrast, the polygyne form contains many fertilized queens per colony, and all three genotypes of Gp-9BB, Gp-9Bb, or Gp-9bb can be present within a colony. Among the study sites, ratios of monogyne:polygyne colonies ranged from 3:55–28:22, and infections rates were 42–78% when both social forms were included in the samples from each site. However, T. solenopsae infections were restricted to colonies of the polygynous social form. While T. solenopsae was only detected in polygynous colonies in the field, T. solenopsae infections were found in ants with the monogyne genotype. Ants from four colonies reared from field-collected, newly mated queens that were naturally infected with T. solenopsae were found to exhibit this genotype. T. solenopsae also was detected in individual alate female reproductives possessing the monogyne genotype, which were collected from polygynous colonies. Polygynous colonies can contain individual ants that possess either the polygyne or monogyne genotype. Thus, T. solenopsae infections can occur in fire ants with genotypes of either social form. Because making genotypic determinations of S. invicta social forms may be impractical in the field, we compared visual and genotypic determinations of polygyny and monogyny. Visual determinations, based mainly on the relative preponderance of major workers, corresponded to 85% of the genotypic determinations.