Single spore derived isolates of Plasmodiophora brassicae are difficult to be generated due to its biotrophic life style. Two methods to produce pure pathotype isolates of P. brassicae were compared. A new technique for the inoculation of Brassica seedlings with single root hairs infected by P. brassicae is described, which is taking advantage of the multiplication of P. brassicae during the first infection cycle in root hairs. Ten single spore derived isolates from 328 single sporangiosorus inoculations were produced using different populations of P. brassicae. To compare success rates inoculations with single resting spores were also made. From 125 inoculations with single resting spores only one single spore derived isolate was achieved. The root hair inoculation method was more practicable than most hitherto reported methods and offers a reasonable efficacy for the production of single spore derived isolates.