Dengue is an emerging mosquito borne disease of public health importance in Nepal. A descriptive cross sectional study was carried out to estimate sero-prevalence and distribution pattern of dengue in certain vulnerable regions of Nepal from June to September 2009. A total of 460 venous blood samples were collected from individuals experiencing a febrile illness clinically consistent with dengue infection visiting nearby hospitals of Kanchanpur, Kailali, Banke, Dang and Chitwan districts. The sero-prevalence of dengue virus specific IgM was determined by enzyme linked immunosorbent assay (ELISA) kit. The anti-dengue IgM positivity was found to be 12.17%. The higher frequency of positive cases (16.4%) were from age group 20-40 years followed by < 20 years age group with 9.7% and 5.3% from > 40 years age group. The association between dengue infection and age is found to be statistically significant (p < 0.05). The male:female ratio was determined as 1.3:1 in IgM positive population. Among sampling areas, Kanchanpur showed highest prevalence of dengue infection (15.5%) followed by Chitwan (11.7%), Kailai (11.1%), Banke (10.7%) and Dang (8.3%). Similarly, 94.6% of the positive cases were indigeneous and had no history of travel to other countries. Dengue is firmly established in terai region with increasing trends of infection and expansion into newer areas raising a public health threat. Regular epidemiological studies are suggested which could further reveal the contributing factors associated with dengue virus infection and help in formulating strategies in reducing the transmission rate and control of the infection.