Cross-amplification permits transposition of microsatellite markers to closely related species. Multiplexing reduces time and cost further, exploiting simultaneous amplification of several primers. In cross-amplification tests of 55 ungulate primers in roe deer, 39 gave specific amplification products and 20 were polymorphic. Twelve primers were retained to form a multiplex kit. In 30 roe deer, average allelic diversity was 5.67 (range: 2–15), expected heterozygosity was 0.664 and mean polymorphic information content (PIC) value was 0.605. Probability of identity was PID= 5 × 10−11 and probability of exclusion for parentage studies was PE1 = 0.98856 and PE2 = 0.99952.