This study collected rectal swabs from diarrheal patients and in-house environmental samples from low-income households in Dhaka City, Bangladesh, over a 4-month period and investigated these to determine the domestic transmission pathways of Escherichia coli-associated diarrhea. The environmental samples included swabs from four frequently touched surfaces, drinking water and food. Both the rectal swabs and environmental samples were examined for virulence genes characteristic of diarrheagenic E. coli pathotypes by PCR. In addition, each sample was cultured for E. coli, and the strains were analyzed for virulence profile and subjected to multilocus sequence typing (MLST). The results showed that 31% (73 of 233) of all samples including rectal swabs and household samples were positive for one or more of the diarrheagenic E. coli virulence factors. PCR analyses showed that 28% (10/36) of the rectal swabs, 43% (58/136) of household swabs, 9% (3/32) of the food, and 7% (2/29) of the water samples were positive for various virulence genes. 6 Out of the 36 rectal swab samples and associated household samples were shown to have similar E. coli pathotypic genes, and the drinking vessel surface was identified as the major source of contamination. EAEC and CTEC were the most commonly identified pathotypes in the cultured isolates. The phylogenetic tree constructed by MLST data showed that the diarrheagenic isolates were clustered in several diversified lineages. This study supports the hypothesis that there are high-risk hotspots, particularly those surfaces associated with food consumption, for diarrheagenic E. coli contamination within the household environments of Bangladesh.