A multiplex PCR method was developed and used to determine the infection rate of Kneallhazia solenopsae in individual Pseudacteon curvatus flies in north-central Florida. Among P. curvatus flies infected with K. solenopsae, 2 amplicons were produced, one of 800 nucleotides from the P. curvatus 18S rRNA gene, and one of 318 nucleotides from the K. solenopsae 16S rRNA gene. Multiplex PCR of DNA extracted from P. curvatus flies was capable of detecting 117.5 ± 82.7 K. solenopsae spore equivalents. The mean K. solenopsae infection rate of P. curvatus from 4 sites in Gainesville and Williston, Florida, was 12.3 ± 5.0%. The K. solenopsae infection rate for P. curvatus was independent of the K. solenopsae infection rate observed among S. invicta nests from where the fly collections took place. Not all P. curvatus flies that developed in K. solenopsae-infected fire ants were positive for K. solenopsae upon eclosion. Among 50 P. curvatus flies known to develop in K. solenopsae-infected S. invicta workers, 12 (24%) were positive for K. solenopsae at eclosion.