Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, both regarding clinical presentation, response to treatment and outcome. Recently, subclassification of DLBCL based on mutational profile has been suggested, and next generation sequencing (NGS) analysis may be relevant as part of the diagnostic workflow. This will, however, often be based on analysis of one tumor biopsy. Here, we present a prospective study where multi-site sampling was performed prior to treatment in patients with newly diagnosed DLBCL. Two spatially different biopsies from 16 patients were analyzed using NGS with an in-house 59-gene lymphoma panel. In 8/16 (50%) patients, mutational differences were found between the two biopsy sites, including differences in TP53 mutational status. Our data indicate that a biopsy from the extra-nodal site may represent the most advanced clone, and an extra-nodal biopsy should be preferred for analysis, if safely accessible. This will help ensure a standardized stratification and treatment decision.