Field evaluations assessing the prevalence of Solenopsis invicta virus 3 (SINV-3) have shown that the virus exhibits a distinct seasonal phenology in the host, Solenopsis invicta, that is negatively correlated with warmer temperatures. Active SINV-3 infections were established in Solenopsis invicta colonies, which were subsequently maintained at 19.1, 22.2, 25.5, 27.7, and 29.3 °C. The quantity of brood declined in all SINV-3-treated colonies regardless of temperature over the initial 30 days. However, the quantity of brood in colonies held at 29.3 °C began increasing (recovering) in the next 40 days until they were statistically equivalent to untreated control colonies. Meanwhile, the quantity of brood continued to decline in colonies held at 19.1, 22.2, 25.5, and 27.7 °C for the duration of the test (81days). By the end of the test, these colonies were in poor health as indicated by decreased brood. Conversely, the amount of brood for colonies held at 29.3 °C increased to above 3, indicating healthy vigorous growth. Worker ants from SINV-3-treated colonies maintained at 19.1, 22.2, and 25.5 °C showed strong production of the VP2 capsid protein by Western blotting; 100% of the colonies sampled (n = 3) showed production of VP2. However, VP2 was detected in only 33% of colonies maintained at 27.7 °C, and the VP2 response was nearly undetectable in all colonies maintained at 29.3 °C. These results indicate that virus assembly does not appear to be occurring efficiently at the higher temperatures. Also, the quantity of SINV-3 detected in queens was significantly lower in those maintained at 29.3 °C compared with the lower temperature treatments. These results indicate that warm summer temperatures combined with fire ant thermoregulatory behavior and perhaps behavioral fevers may explain the low prevalence of SINV-3 in fire ant colonies during the summer.