The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20. Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (Zr) versus non-residualizing (I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation I-GAcDb and I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with I-labeled tracers. Pairwise comparison of Zr- and I-labeled GAcDb and GAcMb allowed assessment of internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells. These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. .