A cross-sectional study was carried out in natural wild rabbit (Oryctolagus cuniculus) populations from southern Spain to identify risk factors associated to myxoma virus infection. Blood samples from 619 wild rabbits were collected, and questionnaires which included variables related to host, disease, game management and environment were completed. A logistic regression analysis was conducted to investigate the associations between myxomatosis seropositivity (dependent variable) across 7 hunting estates and an extensive set of explanatory variables obtained from the questionnaires. The prevalence of antibodies against myxomatosis virus was 56.4% (95% CI: 52.5-60.3) and ranged between 21.4% (95% CI: 9.0-33.8) and 70.2% (95% CI: 58.3-82.1) among the different sampling areas. The logistic regression analysis showed that autumn (OR 9.0), high abundance of mosquitoes (OR 8.2), reproductive activity (OR 4.1), warren's insecticide treatment (OR 3.7), rabbit haemorrhagic disease (RHD) seropositivity (OR 2.6), high hunting pressure (OR 6.3) and sheep presence (OR 6.4) were associated with seropositivity to myxomatosis. Based on the results, diverse management measures for myxomatosis control are proposed.