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Radiolabeling of nanobodies with radiometals by chelation has the advantage of being simple, fast and easy to implement in clinical routine. In this study, we validated 68Ga/111In-labeled anti-VCAM-1 nanobodies as potential radiometal-based tracers for molecular imaging of atherosclerosis. Both showed specific targeting of atherosclerotic lesions in ApoE-/- mice. Nevertheless, uptake in lesions and constitutively VCAM-1 expressing organs was lower than previously reported for the 99mTc-labeled analog. We further investigated the impact of different radiolabeling strategies on the in vivo biodistribution of nanobody-based tracers. Comparison of the pharmacokinetics between 68Ga-, 18F-, 111In- and 99mTc-labeled anti-VCAM-1 nanobodies showed highest specific uptake for 99mTc-nanobody at all time-points, followed by the 68Ga-, 111In- and 18F-labeled tracer. No correlation was found with the estimated number of radioisotopes per nanobody, and mimicking specific activity of other radiolabeling methods did not result in an analogous biodistribution. We also demonstrated specificity of the tracer using mice with a VCAM-1 knocked-down phenotype, while showing for the first time the in vivo visualization of a protein knock-down using intrabodies. Conclusively, the chosen radiochemistry does have an important impact on the biodistribution of nanobodies, in particular on the specific targeting, but differences are not purely due to the tracer's specific activity.