Abstract Purpose Accurate dosimetry is essential in radioembolization. To this purpose, an automatic protocol for healthy liver dosimetry based on dual isotope (DI) SPECT imaging, combining holmium-166 (¹⁶⁶Ho)-microspheres, and technetium-99 m (^99mTc)-colloid was developed: ¹⁶⁶Ho-microspheres used as scout and therapeutic particles, and ^99mTc-colloid to identify the healthy liver. DI SPECT allows for an automatic and accurate estimation of absorbed doses, introducing true personalized dosimetry. However, photon crosstalk between isotopes can compromise image quality. This study investigates the effect of ^99mTc downscatter on ¹⁶⁶Ho dosimetry, by comparing ¹⁶⁶Ho-SPECT reconstructions of patient scans acquired before (¹⁶⁶Ho-only) and after additional administration of ^99mTc-colloid (¹⁶⁶Ho-DI). Methods The ¹⁶⁶Ho-only and ¹⁶⁶Ho-DI scans were performed in short succession by injecting ^99mTc-colloid on the scanner table. To compensate for ^99mTc downscatter, its influence was accounted for in the DI image reconstruction using energy window-based scatter correction methods. The qualitative assessment was performed by independent blinded comparison by two nuclear medicine physicians assessing 65 pairs of SPECT/CT. Inter-observer agreement was tested by Cohen’s kappa coefficient. For the quantitative analysis, two volumes of interest within the liver, VOI_TUMOR, and VOI_HEALTHY were manually delineated on the ¹⁶⁶Ho-only reconstruction and transferred to the co-registered ¹⁶⁶Ho-DI reconstruction. Absorbed dose within the resulting VOIs, and in the lungs (VOI_LUNGS), was calculated based on the administered therapeutic activity. Results The qualitative assessment showed no distinct clinical preference for either ¹⁶⁶Ho-only or ¹⁶⁶Ho-DI SPECT (kappa = 0.093). Quantitative analysis indicated that the mean absorbed dose difference between ¹⁶⁶Ho-DI and ¹⁶⁶Ho-only was − 2.00 ± 2.84 Gy (median 27 Gy; p value < 0.00001), − 5.27 ± 8.99 Gy (median 116 Gy; p value = 0.00035), and 0.80 ± 1.08 Gy (median 3 Gy; p value < 0.00001) for VOI_HEALTHY, VOI_TUMOR, and VOI_LUNGS, respectively. The corresponding Pearson’s correlation coefficient between ¹⁶⁶Ho-only and ¹⁶⁶Ho-DI for absorbed dose was 0.97, 0.99, and 0.82, respectively. Conclusion The DI protocol enables automatic dosimetry with undiminished image quality and accuracy. Clinical trials The clinical study mentioned is registered with Clinicaltrials.gov (NCT02067988) on 20 February 2014.