Until now, molecular and biochemical methods have only been used to show whether or not Plasmodiophora brassicae is present in plant or soil samples but not to what extent. Here, in planta quantification of P. brassicae by whole-cell fatty acid (WCFA) measurements and real-time polymerase chain reaction (PCR) was evaluated. Arachidonic acid (ARA, 20:4) was the most abundant fatty acid in resting spores and was only found in infected roots, which indicates a potential of ARA as a biomarker for P. brassicae. A real-time PCR assay was developed using primers designed from the internal transcribed spacer region of the ribosomal DNA. Using these primers, it was possible to detect P. brassicae in infected roots 10 days after germination of plants sown in infested soil. A bioassay showed that the amounts of ARA found by WCFA analysis and the DNA found by real-time PCR in infected plants were well correlated. These measurements also correlated with the soil spore content and the assessed disease incidence and disease severity scores. Therefore, we conclude that WCFA analysis and real-time PCR are good tools for P. brassicae quantification that can be applied to basic studies of the pathogen and in resistance screens.