BackgroundVector-borne infections pose significant health risks to humans, domestic animals, and wildlife. Domestic dogs (Canis lupus familiaris) in the United States may be infected with and serve as sentinel hosts for several zoonotic vector-borne pathogens. In this study, we analyzed the geographical distribution, risk factors, and co-infections associated with infection withEhrlichiaspp.,Anaplasmaspp.,Borrelia burgdorferi, andDirofilaria immitisin shelter dogs in the Eastern United States.MethodsFrom 2016 to 2020, blood samples from 3750 shelter dogs from 19 states were examined with IDEXX SNAP®4Dx®Plus tests to determine the seroprevalence of infection with tick-borne pathogens and infection withD. immitis. We assessed the impact of factors including age, sex, intact status, breed group, and location on infection using logistic regression.ResultsThe overall seroprevalence ofD. immitiswas 11.2% (n= 419/3750), the seroprevalence ofAnaplasmaspp. was 2.4% (n= 90/3750), the seroprevalence ofEhrlichiaspp. was 8.0% (n= 299/3750), and the seroprevalence ofB. burgdorferiwas 8.9% (n= 332/3750). Regional variation in seroprevalence was noted:D. immitis(17.4%,n= 355/2036) andEhrlichiaspp. (10.7%,n= 217/2036) were highest in the Southeast while seroprevalence forB. burgdorferi(19.3%,n= 143/740) andAnaplasmaspp. (5.7%,n= 42/740) were highest in the Northeast. Overall, 4.8% (n= 179/3750) of dogs had co-infections, the most common of which wereD. immitis/Ehrlichiaspp. (1.6%,n= 59/3750),B. burgdorferi/Anaplasmaspp. (1.5%,n= 55/3750), andB. burgdorferi/Ehrlichiaspp. (1.2%,n= 46/3750). Risk factors significantly influenced infection across the evaluated pathogens were location and breed group. All evaluated risk factors were significant for the seroprevalence ofD. immitisantigens.ConclusionsOur results demonstrate a regionally variable risk of infection with vector-borne pathogens in shelter dogs throughout the Eastern United States, likely due to varying distributions of vectors. However, as many vectors are undergoing range expansions or other changes in distribution associated with climate and landscape change, continued vector-borne pathogen surveillance is important for maintaining reliable risk assessment.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05813-1.