Visceral leishmaniasis is one of the most important parasitic diseases that is endemic in some parts of Iran. This study aimed to determine current distribution of visceral leishmaniasis in four distinct geographical zones of Iran. A cross-sectional study was conducted using direct agglutination test (DAT) on 9396 and 2559 serum samples collected from humans and domestic dogs, respectively during the period of 2007 through 2009. Altogether, 403 (4.3%) out of 9396 human serum samples collected from 4 distinct geographical locations showed anti-Leishmania antibodies with titers ≥ 1:3200. Physical examinations performed on 142 sero-positive cases with anti-Leishmania antibodies at titers of 1: 3200 to 1:102400 among whom fever (94.4%), paleness (67.6%) and hepato-splenomegaly (42.2%) were the predominant clinical signs and symptoms. The highest sero-prevalence rate (1.55%) was found in children ≤ 5 years old. Out of 2559 serum samples collected from domestic dogs, 212 (8.3%) were DAT positive (≥ 1:320). Leishmania infantum is the principal causative agent of the disease was isolated from both infected humans and dogs in Iran. Our findings indicate that Mediterranean visceral leishmaniasis with different distribution occurs in different geographical locations of Iran.