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Entamoeba histolytica is believed to be a ubiquitous cause of pediatric non-bloody diarrhea. Using a multiplex polymerase chain reaction assay, we identified stool pathogens in 298 Guatemalan children presenting with acute, non-bloody diarrhea. Despite a high overall pathogen burden, we identified no cases of E. histolytica. Knowledge of local epidemiology is important to inform E. histolytica therapeutic strategies in low-income settings.