A sample of pure Terbium-161 with radioactivity over 1 GBq was prepared at the Faculty of Nuclear Sciences and Physical Engineering, CTU in Prague thanks to a newly developed separation system. It was prepared by neutron irradiation of highly enriched Gadolinium-160 and subsequent separation on a chromatographic column with cation exchange resin Dowex 50W×8(H+) (100-200 mesh) using alpha-HIBA as eluent. After separation, Tb-161 containing fractions were purified on another Dowex column to eliminate alpha-HIBA. Pure Tb-161 fractions were obtained by elution with 1M and 4M HCl in several fractions. Total amount of Tb-161 obtained after separation was 5925,3 MBq (related to the end of beam). Radionuclide purity of prepared sample reached 99,9996% and specific Tb-161 activity of one of fractions reached 40 GBq/µg Tb.