Siesta Key Beach, located on the Gulf Coast of Florida, is frequently mentioned among the top ten beaches in the US. In summer 2004, high levels of indicator bacteria caused health warnings to be posted, and a storm drainage system was implicated as a possible source of microbial contamination. A study was initiated to determine whether indicator bacteria that persisted in the stormwater system could contribute to high microbial loads in receiving waters. Two sampling events, one within 48 hours of a rain event and the other during dry conditions, were conducted. Water and sediment samples were taken at various sites from the storm drainage system to the beach. Fecal coliforms and Enterococcus spp. were enumerated, and genotypic fingerprints of E. coli and Enterococcus spp. were generated by BOX-PCR. Diversity of E. coli and Enterococcus populations was calculated with the Shannon-Weiner diversity index. Similarity of E. coli and Enterococcus populations was calculated with the population similarity coefficient. After the rain event, levels of fecal coliforms and Enterococcus spp. were high in sediments and exceeded the regulatory standard for all water samples. In dry conditions, levels were lower in water samples, but still high in sediment samples. Significantly greater population diversity was observed in the rain event compared to the dry event for both E. coli and Enterococcus populations, and greater population similarity was vi observed in dry conditions. Enterococcus population diversity was significantly higher in untreated sewage and the Siesta Key rain event when compared to dry conditions, and to a site on the Myakka River (no known human input or urban stormwater runoff). Siesta Key populations in dry conditions were most similar to Myakka, and sewage was the least similar to all other populations. Increased population similarity for E. coli and Enterococcus spp. during dry conditions suggests that a portion of the population is composed of “survivor” isolates. Persistence of survivor isolates in the storm drainage system, where urban runoff can sit for days, suggests a reservoir for indicator bacteria that can be flushed through the system to the Gulf, causing high levels of indicator bacteria in receiving waters.