Increasing risk factors are making leishmaniases a growing public health concern for many countries around the world. The aim of this study was to assess the seroprevalence of Leishmania infantum infection in the general population and in HIV infected subjects of Northern Greece, bordering the Mediterranean basin where leishmaniasis is endemic. The clinical cases of the disease during the last 6 years (2001-2006) are also presented. A low frequency of L. infantum antibodies was found by IFA and ELISA in 1,525 healthy individuals (2.8%), aged 18-80 years, living in the 16 prefectures of Northern Greece (Macedonia and Thrace regions), and in 167 HIV positive subjects (0.6%). Fifty-seven clinical cases were diagnosed in the same area and an approximate annual incidence of 0.34/100,000 was estimated. No endemic foci were identified and the cases of the disease were sporadic. Most presented with the visceral form (VL), few with the cutaneous, and one with VL-HIV co-infection. A significant shift in the age of people at risk was observed, with children less affected than adults (children/adults ratio: 0.36). No relevant data from previous studies are available to demonstrate a possible change of the infection in Northern Greece. The results of this study could be used as a reference for leishmaniasis surveillance in the area.