BACKGROUND: Lyme borreliosis is a worldwide zoonotic disease caused by spirochetes of the Borrelia burgdorferi sensu lato complex. There are no reports on this subject in dogs from Iran. OBJECTIVES: Determining the serologic prevalence level of produced antibodies against Borrelia burgdorferi sensu lato complex in three Caspian littoral provinces of Iran and studying the effect of climatic risk factors on it are the first aims of this study. METHODS: During the period from July to September 2009 a seroepidemiological study was conducted on 273 dogs in three Caspian provinces of Guilan, Mazandaran and Golestan, Iran's known habitats of tick (Ixodes ricinus). In order to study the correlation between infection distribution and climatic factors by geographic information system (GIS), geographic position of seronegative and seropositive dogs was overlaid on climatic maps of Guilan, Mazandaran and Golestan provinces. Multivariate regression model and correlation matrix analyses were used for statistical analysis. RESULTS: From 273 serum samples in the whole studied area, 22 (8.1%) showed antibodies against B. burgdorferi sensu lato complex. The seroprevalence of B. burgdorferi sensu lato in provinces of Guilan, Mazandaran and Golestan were 0.0% (0.91), 2.2% (2.91) and 22% (20.91), respectively. Mean annual temperature had positive and significant correlation with B. burgdorferi sensu lato complex seroprevalence in sampled dogs of the three north provinces (p<0.05). CONCLUSIONS: Regarding the seroprevalence of Lyme borreliosis in dogs of three Caspian provinces of Iran, more attention must be paid to this disease, especially in Golestan province. This is the first study on the role of climatic factors in canine Lyme borreliosis in Iran.