Visceral leishmaniasis (VL) caused byLeishmania donovaniis endemic in northern Ethiopia, whereP. orientalisis the most important presumed vector. This study was designed to determine the physiological age structure and the occurrence ofLeishmaniainfection in the vector of VL in Tahtay Adiyabo district, northern Ethiopia. Sand flies were collected using CDC light traps from peridomestic and agricultural fields between May 2011 and April 2012 andP. orientalisfemales were dissected for age determination and detection ofLeishmaniapromastigotes. Sand flies were also analyzed forL. donovanidetection using molecular methods. Of 1,282P. orientalisexamined for abdominal stages and age characterization, 66.2%, 28.2%, 4.1%, and 1.6% were unfed, freshly fed, half-gravid, and gravid. Parous rate in unfed females was 34.1% and 35.4% in peridomestic and agricultural fields, respectively. Out of 921P. orientalisfemales dissected, one specimen (0.1%) was found naturally infected with promastigotes. Five pools (25 females) of unfedP. orientaliswere also found with DNA ofLeishmaniaspp. In particular, a singleP. orientaliswas positive forL. donovani(0.5%). Based on this and other evidences (abundance, human blood feeding, and xenodiagnostic studies),P. orientalisis the principal vector of VL in this endemic focus.