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The red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), originally is from South America and currently infests over 128 million ha in the US. Its presence has caused significant social, environmental, and economic impacts. Over the decades, chemical insecticides have controlled these pest ants successfully. However, this method is costly and unsustainable because red imported fire ant re-establishes colonies quickly after chemical application. Thus, it is important to develop additional strategies for managing the red imported fire ant in the US. Solenopsis invicta virus 3 is a positive-sense, single-stranded RNA virus specific for S. invicta that offers promise as a classical biological control agent or biopesticide for control of S. invicta. Surveys were conducted to determine the prevalence of Solenopsis invicta virus 3 in alates of S. invicta collected from 5 urban areas (Tallahassee, Pensacola, Jacksonville, Gainesville, and Panama City) and 5 adjacent rural areas (Quincy, Jay, Macclenny, Lake City, and Blountstown) of North Florida, USA, using the reverse transcription polymerase chain reaction technique. The prevalence of Solenopsis invicta virus 3 varied widely from city to city. No statistically significant differences in alate infection rate was found between urban and rural cities sampled. Areas in which no infections of Solenopsis invicta virus 3 were detected may be good candidates for the introduction of this virus as a biological control agent to help manage this pest locally. [ABSTRACT FROM AUTHOR]