Objectives To determine whether pretreating diagnostic samples with heat increases the detection of Dirofilaria immitis antigen in adult cats, we evaluated feline serum and plasma samples collected in heartworm-endemic areas of the southern United States. Methods Commercial microtiter well assays for detection of D immitis antigen were used to evaluate serum or plasma samples from 385 shelter and free-roaming cats from the southcentral and southeastern United States before and after heat treatment; commercial antibody tests were performed on a subset of samples. Results Prior to sample heat treatment, 1/220 (0.5%) shelter cats and 4/165 (2.4%) free-roaming cats had detectable D immitis antigen. After heat pretreatment, the detection rate increased to 13/220 (5.9%) and 13/165 (7.9%), respectively. Antibody reactive to D immitis was significantly more common ( P <0.001) in the serum of cats that were antigen positive after heat treatment (10/13; 76.9%) than serum from cats that remained antigen negative after heat treatment (22/163; 13.5%). Conclusions and relevance Heat pretreatment of feline samples increased antigen detection by commercial assays for D immitis and improved overall concordance of antigen and antibody test results in antigen-positive samples in this population. Although further work to investigate the specificity of D immitis antigen assays when using pre-treated samples is warranted, this approach may be useful in the diagnosis of heartworm infection in individual cats and may increase the accuracy of surveys based on antigen detection.