Toxoplasma gondii is a globally distributed parasite infecting humans and warm-blooded animals. Although many surveys have been conducted for T. gondii infection in mammals, little is known about the detailed distribution in localized natural populations. In this study, host genotype and spatial location were investigated in relation to T. gondii infection. Wood mice (Apodemus sylvaticus) were collected from 4 sampling sites within a localized peri-aquatic woodland ecosystem. Mice were genotyped using standard A. sylvaticus microsatellite markers and T. gondii was detected using 4 specific PCR-based markers: SAG1, SAG2, SAG3 and GRA6 directly from infected tissue. Of 126 wood mice collected, 44 samples were positive giving an infection rate of 34·92% (95% CI: 27·14–43·59%). Juvenile, young adults and adults were infected at a similar prevalence, respectively, 7/17 (41·18%), 27/65 (41·54%) and 10/44 (22·72%) with no significant age-prevalence effect (P = 0·23). Results of genetic analysis of the mice showed that the collection consists of 4 genetically distinct populations. There was a significant difference in T. gondii prevalence in the different genotypically derived mouse populations (P = 0·035) but not between geographically defined populations (P = 0·29). These data point to either a host genetic/family influence on parasite infection or to parasite vertical transmission.