Introduction: For stratification of chemotherapeutic choices, radiopharmaceuticals capable of imaging breast cancer resistance protein (BCRP/ABCG2)-mediated functional transport are desired. To accomplish this objective, Galmydar, a fluorescent and moderately hydrophobic Ga(III) cationic complex and its 67/68Ga-radiolabeled counterparts were interrogated in HEK293 cells stably transfected with BCRP and their WT counterparts transfected with empty vector. Additionally, the sensitivity and specificity of 68Ga-Galmydar to evaluate functional expression of BCRP at the blood-brain barrier (BBB) was investigated in gene-knockout mdr1a/1b(-/-) (double knockout, dKO) and mdr1a/1b(-/-)ABCG2(-/-) (triple knockout, tKO) mouse models. Methods: For radiotracer uptake assays and live cell fluorescence imaging, either 67Ga-Galmydar or its unlabeled counterpart was incubated in HEK293 cells transfected with BCRP (HEK293/BCRP) and their WT counterparts at 37 °C under a continuous flux of CO2 (5%) in the presence or absence of Ko143, a potent BCRP antagonist, and cellular uptake was measured to assess the sensitivity of Galmydar to probe BCRP-mediated functional transport activity in cellulo. For assessing the potential of Galmydar to enable diagnostic imaging of targeted tissues in vivo, the 67Ga-radiolabeled counterpart was incubated in either human serum albumin or human serum at 37 °C and the percentage of unbound 67Ga-Galmydar was determined. To evaluate the sensitivity of 68Ga-Galmydar for molecular imaging of BCRP-mediated efflux activity in vivo, microPET/CT brain imaging was performed in dKO and tKO mice and their age-matched WT counterparts, 60 min post-intravenous injection. Results: 67Ga-Galmydar shows uptake profiles in HEK293 cells inversely proportional to BCRP expression, and antagonist (Ko143) induced accumulation in HEK293/BCRP cells, thus indicating target sensitivity and specificity. Furthermore, employing the fluorescent characteristics of Galmydar, optical imaging in HEK293/BCRP cells shows an excellent correlation with the radiotracer cellular accumulation data. 67Ga-Galmydar shows > 85% unbound fraction and presence of parental compound in human serum. Finally, microPET/CT imaging shows higher retention of 68Ga-Galmydar in brains of dKO and tKO mice compared to their age-matched WT counterparts, 60 min post-intravenous tail-vein injection. Conclusions: Combined data indicate that Galmydar could provide a template scaffold for development of a PET tracer for imaging BCRP-mediated functional transport activity in vivo.