Background: Leishmaniasis is primarily caused by two species of Leishmania (L. donovani and L. infantum) of which clinical infection with L. infantum has been recognized in both humans and dogs as zoonotic disease with dogs as the main reservoir hosts in the Mediterranean, the Middle East, Asia and South America. Although L. donovani has been associated with both clinical and asymptomatic infection in humans but it is still associated with asymptomatic infection in dogs in Indian sub-continent without any evidence of zoonotic infection. Objectives: The objective of this research was to investigate the potentiality of dog as reservoir host for visceral leishmaniasis in the human leishmaniasis endemic regions in Bangladesh. Materials and Methods: A total of 20 stray dogs in the human VL endemic areas of Mymensingh district were captured for the detection VL during the period of November 2010 to May 2011. The dipstick test rK39 (Bios International; n = 20), Giemsa’s stained impression smears of liver and spleen (n = 6) and PCR with the tissue of liver and spleen (n = 6) were tested as per manufacturer instructions and conventional standard methods. Results: Out of 20 stray dogs examined, 4 (20.0%) were positive for L. donovani infection with rK39 strip test. Of the six randomly selected dogs tested with Modified Giemsa’s stained of impression smears of spleen and liver showed 2 (33.33%) positive whereas PCR technique detected 5 (83.33%) positive for L. donovani. Results of PCR showed 145bp amplicon, specific for L. donovani infection in 83.33% stray dogs. Conclusions: This study reveals that a high percentage of L. donovani asymptomatic carrier infections occur in dogs and evidence indicates that dogs and humans may potentially serve as a source of infection to sand fly vectors and accordingly dogs can be recognized as a probable animal reservoir for the Leishmania infection in the endemic region in Bangladesh. However, further studies are required to determine the ability of dogs to transmit the L. donovani to the vector sand fly in nature and its evidence on ‘One Health’ perspectives. Keywords: Visceral leishmaniasis, Endemic region, Stray dogs, rK39 strip test, Giemsa’s stained liver and spleen impression smears, PCR, Reservoir host, Mymensingh