Oligonucleotide primer pairs were designed to unique areas of the small subunit (16S) rRNA gene of Thelohania solenopsae and a region of the Gp-9 gene of Solenopsis invicta. Multiplex PCR resulted in sensitive and specific detection of T. solenopsae infection of S. invicta. The T. solenopsae-specific primer pair only amplified DNA from T. solenopsae and T. solenopsae-infected S. invicta. This primer pair did not produce any amplification products from DNA preparations from uninfected S. invicta, seven additional species of microsporidia (including Vairimorpha invictae), or Mattesia spp. The Gp-9-specific primers recognized and amplified DNA from Solenopsis xyloni, Solenopsis richteri, Solenopsis geminata, the invicta/richteri hybrid, and monogyne and polygyne S. invicta, but not from T. solenopsae, and, as such, served as a positive control verifying successful DNA preparation. Multiplex PCR detected T. solenopsae in worker fire ants infected with as few as 5000 spores. Furthermore, multiplex PCR detected T. solenopsae in all developmental stages of S. invicta. However, detection could be made more sensitive by using only the T. solenopsae-specific primer pair; ants infected with as few as 10 spores were able to be discerned. Multiplex PCR detection of T. solenopsae offers the advantages of a positive control, a single PCR amplification, detection of all developmental stages, and increased sensitivity and specificity compared with microscopy.