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The primary objective of this study was to determine the microbial water quality of a large irrigation system and how this quality varies with respect to canal size, impact of near-by communities, and the travel distance from the source in the El Valle del Yaqui, Sonora, Mexico. In this arid region, 220,000 hectares are irrigated with 80% of the irrigation water being supplied from an extensive irrigation system including three dams on the Yaqui River watershed. The stored water flows to the irrigated fields through two main canal systems (severing the upper and lower Yaqui Valley) and then through smaller lateral canals that deliver the water to the fields. A total of 146 irrigation water samples were collected from 52 sample sites during three sampling events. Not all sites could be accessed on each occasion. All of the samples contained coliform bacteria ranging from 1,140 to 68,670 MPN/100 mL with an arithmetic mean of 11,416. Ninety-eight percent of the samples contained less than 1,000 MPN/100 mL Escherichia coli, with an arithmetic mean of 291 MPN/100 mL. Coliphage were detected in less than 30% of the samples with an arithmetic average equal to 141 PFU/100 mL. Enteroviruses, Cryptosporidium oocysts, and Giardia cysts were also detected in the canal systems. No significant difference was found in the water quality due to canal system (upper or lower Yaqui Valley), canal-size (main vs. lateral), distance from source, and the vicinity of human habitation (presence of various villages and towns along the length of the canals). There was a significant decrease in coliforms (p < 0.011) and E. coli (< 0.022) concentrations as travel distance increased from the City of Obregon.