The dramatic decline of the native red squirrel in the UK has been attributed to both direct and disease-mediated competition with the grey squirrel where the competitor acts as a reservoir host of squirrelpox virus (SQPV). SQPV is threatening red squirrel conservation efforts, yet little is known about its epidemiology. We analysed seroprevalence of antibody against SQPV in grey squirrels from northern England and the Scottish Borders in relation to season, weather, sex, and body weight using Generalized Linear Models in conjunction with Structural Equation Modelling. Results indicated a heterogeneous prevalence pattern which is male-biased, increases with weight and varies seasonally. Seroprevalence rose during the autumn and peaked in spring. Weather parameters had an indirect effect on SQPV antibody status. Our findings point towards a direct disease transmission route, which includes environmental contamination. Red squirrel conservation management options should therefore seek to minimize squirrel contact points.