Thelohania solenopsae, a microsporidian parasite of Solenopsis invicta, the Red Imported Fire Ant (RIFA), is studied as a potential biocontrol agent. Here, we report the patterns of occurrences of T. solenopsae in the populations of RIFA in Oklahoma. As a long-term study, RIFA samples were collected and analyzed twice a year, since fall 2002, from two sites at Bryan County and one site at McCurtain County. Although earlier attempts to release and establish this pathogen on RIFA population in Oklahoma had failed, T. solenopsae have been detected in the samples of S. invicta collected at Bryan County since 2002. These occurrences were confirmed by PCR and modified Trichrome staining. The RIFA samples tested positive for T. solenopsae at varied proportions in the two sites at Bryan County ranging from 85% to 90% during fall 2002 to about 30%-33% in spring 2003 and 47%-65% in fall 2003 to 54% to 65% in spring 2004. The RIFA population in McCurtain County was devoid of the microsporidian parasite until recently. RIFA samples collected within 35 miles along the highway from Bryan County were infected with T. solenopsae and none beyond up to McCurtain County. These patterns in the occurrence of the parasite in the fire ant population indicate that T. solenopsae is in the process of establishing in RIFA colonies. Further understanding of the patterns of occurrence of T. solenopsae in the fire ants will be monitored by highway sampling and by long-term studies of the two sites at Bryan County.