Escherichia coli, a prominent waterborne pathogen, causes a variety of gastrointestinal and extraintestinal infections that depend on virulence determinants. To monitor natural aquatic systems for virulence-associated genes of E. coli, multiplex PCR was used in a survey covering 46 major natural water bodies in Bangladesh. DNA was extracted directly from water samples as well as from pre-enriched and enriched cultures during three successive seasons and assessed for E. coli virulotype distribution. From the five virulotypes, genes from the enterotoxigenic (ETEC), enteropathogenic (EPEC), and enterohaemorrhagic (EHEC) virulotypes were detected consistently, but genes from the enteroinvasive (EIEC) and enteroaggregative (EAEC) virulotypes were traced only occasionally. ETEC was the most prevalent virulotype, followed by EPEC. However, EIEC and EAEC virulotypes could not be detected in winter or the rainy season, respectively. Specific regional distribution patterns of different E. coli virulotypes and their temporal fluctuations were identified. These observations may assist with assessing seasonal risk and identifying vulnerable areas of the country prone to E. coli-associated outbreaks.