The natural reservoirs for avian influenza virus (AIV) are wild bird species of the orders Anseriformes and Charadriiformes. The primary route of transmission for wild birds is through fecally contaminated surface water on shared aquatic habitats. A distilled water model has shown that AIV remains infectious in water for weeks to months with pH, salinity, and temperature affecting stability. To evaluate the effect of pH, salinity, and temperature on AIV persistence in natural surface water, we measured the duration of infectivity for two common low pathogenic AIV subtypes in 15 filtered surface water samples collected from major waterfowl habitats in Georgia, USA. Trials were performed at three incubation temperatures 10, 17, and 28 C. Consistent with previous studies, pH and temperature had a significant effect on the stability of AIV in filtered surface water. Both viruses were less stable at warmer temperatures and in acidic water (pH <5.0). Due to the limited range of salinity of the field water samples, the role of salinity in AIV stability in surface water could not adequately be evaluated. Variations in persistence times between water samples with comparable pH and salinities indicated that other factors affect AIV stability in natural surface water. These results contribute to the current understanding of AIV persistence in aquatic habitats and may help in identifying areas with an increased likelihood of AIV persistence and potential transmission.