Agricultural by-products are becoming an attractive substrate for bioethanol production. The aim of this study was to evaluate the effects of regional differences in the rice hulls using Escherichia coli KO11 for bioethanol production. The rice hulls coded Edirne were obtained from Thrace Region, and the rice hulls coded Izmir were obtained from Aegean Region in Turkey. Rice hulls were treated by dilute acid before using them as substrates. The cells were incubated on an orbital shaker at 160 rpm under 30 °C during 96 h of the fermentation period. It was found that the maximum yield of ethanol from sugar (0.44 g ethanol/g reducing sugar) was obtained with the substrate C/N ratio of 29.16 in Izmir medium. The main difference was the dominant carbon source available as a substrate. It was detected that glucose concentration was about 2.5 times higher in Izmir medium, whereas xylose concentration was about two times higher in Edirne medium. The different results obtained with rice hulls from different origins could depend on the type of paddy as well as different cultivation conditions. These findings provide a valuable indicator for identifying suitable agricultural waste materials to be used as substrates for bioethanol production.