Purpose <p>Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an attractive molecular target for anti-tumor therapies undergoing late-stage clinical development. Quantitative imaging of ROR1 expression could enable identification of patients likely to respond to such treatments. This study aimed to assess the feasibility of specific <i>in vivo</i> imaging of ROR1 using radiolabeled affibody molecules.</p> Methods <p>The affibody molecule ZROR1:A10, with high affinity for human (4&#xa0;nM) and murine (2&#xa0;nM) ROR1, was labeled with indium-111 using a DOTA chelator. <i>In vitro</i> binding specificity, affinity, and cellular processing were evaluated using a panel of ROR1-expressing cell lines. The affibody [¹¹¹In]In-DOTA-Zcov19s, which does not bind ROR1, served as a nonspecific control. The biodistribution of [¹¹¹In]In-DOTA-ZROR1:A10 and [¹¹¹In]In-DOTA-Zcov19s was measured in immunodeficient mice bearing ROR1-positive tumors, with ROR1-negative Ramos xenografts used as an additional specificity control.</p> Results <p>[¹¹¹In]In-DOTA-ZROR1:A10 bound specifically to ROR1-expressing cells, with significantly higher uptake than [¹¹¹In]In-DOTA-Zcov19s. The apparent equilibrium dissociation constant for ROR1 binding <i>in vitro</i> was 1–4&#xa0;nM. Tumor uptake of [¹¹¹In]In-DOTA-ZROR1:A10 in MDA-MB-468 xenografts was saturable and significantly (p &lt; 0.05) higher than in Ramos tumors, as well as higher than uptake of the control affibody. At 4&#xa0;h post‑injection of 1&#xa0;µg (50&#xa0;µg/kg), tumor‑to‑blood, tumor‑to‑bone, and tumor‑to‑muscle ratios were 14 ± 6, 19 ± 6, and 11 ± 1, respectively. Micro-SPECT/CT imaging clearly visualized ROR1‑positive tumors and discriminated them from ROR1‑negative xenografts.</p> Conclusion <p>The preclinical evaluation shows that visualization of ROR1-expressing tumors using a radiolabeled affibody molecule is feasible.</p>