Description: Antibodies have become an established treatment modality in cancer therapy during the last decade. However, these treatments often suffer from an insufficient and heterogeneous response despite validated antigen or target receptor expression in the tumor. In fact, therapeutic success depends on both the presence of the tumor antigen and its accessibility by the antibody. In search of a suitable preclinical animal model to evaluate the mechanisms of tumor heterogeneity and hemodynamics, we characterized two exemplary non-Hodgkin lymphoma subtypes with comparable CD20 expression and metabolism, SUDHL-4 and Granta-519, using multimodal imaging techniques. Methods: To investigate in vivo biodistribution, two differently modified αCD20 antigen-binding fragments (Fab), prepared by PASylation with a 200-residue polypeptide tag comprising Pro, Ala, and Ser (PAS 200 ) and by fusion with an albumin-binding domain (ABD), were radiolabeled with 125 I and intravenously injected into immunocompromised mice bearing corresponding xenografts. Results: Validation with 18 F-FDG revealed a similar distribution in vital tumor tissue 1 h after injection. However, large differences in tumor uptake were observed when the CD20-specific radiotracers 125 I-Fab-ABD and 125 I-Fab-PAS 200 were applied (respective percentages injected dose per gram at 24 h after injection: 12.3 and 2.4 for Granta-519 vs. 5.8 and 1.2 for SUDHL-4). Three-dimensional light-sheet fluorescence microscopy with Cy5-Fab-PAS 200 confirmed better tracer extravasation in the Granta-519 tumors. Moreover, dynamic contrast-enhanced (DCE) MRI revealed significantly reduced perfusion in the SUDHL-4 tumors. Conclusion: Tracer uptake was highly dependent on local tumor perfusion and Fab permeation in the SUDHL-4 and Granta-519 tumors. Thus, the SUDHL-4 xenograft offers an excellent model for investigating the influence of therapies affecting tumor angiogenesis.