Diarrhea is one of the leading public health problems in under developed countries. In Pakistan, diarrheal cases are treated empirically and, rarely, specific investigations are made. This study was designed to identify the prevalent strains of Escherichia coli among children of less than five years of age in Quetta, Balochistan, Pakistan. A total of 200 samples were collected from children suffering with diarrhea. E. coli was isolated using standard biological procedures and confirmed by colonial characteristics, biochemical profile and specie specific PCR. Multiplex PCR was applied to differentiate among the five main diarrheagenic E. coli pathotypes (enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC). Results show that the virulent EAEC strain of E. coli was found to be the most prevalent (35%), while EPEC was identified in 15%, followed by EHEC (8%). Notably, none of the samples was found positive for EIEC. Moreover, ETEC was identified in 11% cases of co-infection. Results showed that children with less than two years of age were affected significantly. However, gender had no significant effect on acquiring diarrhea. In conclusion, pathogen spectrum of pathogenic E. coli is EAEC strain. Children under one year of age were affected significantly, and no significant effect was observed when data was stratified on gender base. Further studies are required to investigate the other pathological agents and control parameters should be strengthened to minimize the risks.