An expression library was created and 2304 clones sequenced from a monogyne colony of Solenopsis invicta. The primary intention of the project was to utilize homologous gene identification to facilitate discovery of viruses infecting this ant pest that could potentially be used in pest management. Additional genes were identified from the ant host and associated pathogens that serve as an important resource for studying these organisms. After assembly and removal of mitochondrial and poor quality sequences, 1054 unique sequences were yielded and deposited into the GenBank database under Accession Nos. EH412746 through EH413799. At least nine expressed sequence tags (ESTs) were identified as possessing microsatellite motifs and 15 ESTs exhibited significant homology with microsporidian genes. These sequences most likely originated from Thelohania solenopsae, a well-characterized microsporidian that infects S. invicta. Six ESTs exhibited significant homology with single-stranded RNA viruses (3B4, 3F6, 11F1, 12G12, 14D5, and 24C10). Subsequent analysis of these putative viral ESTs revealed that 3B4 was most likely a ribosomal gene of S. invicta, 11F1 was a single-stranded RNA (ssRNA) virus contaminant introduced into the colony from the cricket food source, 12G12 appeared to be a plant-infecting tenuivirus also introduced into the colony as a field contaminant, and 3F6, 14D5, and 24C10 were all from a unique ssRNA virus found to infect S. invicta. The sequencing project illustrates the utility of this method for discovery of viruses and pathogens that may otherwise go undiscovered.