Visceral leishmaniasis (VL) caused by is a public health threat in the Emilia-Romagna region, northeastern Italy, but its epidemiology has not been fully elucidated in this area. The objective of this study was to characterize infection in sand flies collected in a re-emerging focus of VL in the Bologna province. During the summer of 2016, 6114 sand flies were collected, identified, and tested for detection. Of the identified sand flies, 96.5% were and 3.5% were . Detected parasites were characterized by biomolecular methods (multilocus microsatellite typing and characterization of repetitive region on chromosome 31), and quantified by real-time PCR. The prevalence of infection in individually-tested sand flies varied from 6% to 10% with an increasing trend during the season. Promastigotes of were isolated by dissection in one female the isolated strain (Lein-pw) were closely related to parasites from VL cases in northeastern Italy, but differed from strains isolated in dogs from the same area. Our findings strongly support the vector status of for human VL in the study area.