INTRODUCTION: Tumor mutational burden (TMB) is an emerging biomarker used to identify patients more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, biological variables like tumor cell content and intratumor heterogeneity (ITH) may play an important role in determining TMB. METHODS: TMB estimates were determined applying the TruSightTM Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multi-region sequencing (2-6 samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in 5 patients. RESULTS: On average, a coding region of 1.28 Mbp was covered with a mean read depth of 609x. Manual validation of the mutation-calls confirmed a good performance, but revealed noticeable misclassification during germline filtering. Different regions within a tumor showed considerable spatial TMB variance in 30% (7/24) of the cases (max difference 14.13 mut/Mbp). Lymph node derived TMB was significantly lower (p=0.016). In 13 cases, distinct mutational profiles were exclusive to different regions of a tumor, leading to higher values for simulated aggregated TMB. Combined, ITH and the aggregated TMB could result in divergent TMB designation in 17% of the analyzed patients. TMB variation between primary tumor and distant metastases existed but was not profound. CONCLUSIONS: Our data demonstrate that, in addition to technical aspects like germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of single sample based TMB estimations in a clinical context.