Real-time PCR assays for Colletotrichum acutatum, one of the most important pathogens of strawberry worldwide, were developed using primers designed to the ribosomal DNA internal transcribed spacer 1 ( rDNA ITS1) and the beta-tubulin 2 gene. Using TaqMan technology, the ITS-based assay could reliably detect as little as 50 fg genomic DNA, 100 copies of target DNA, or 25 conidia. The beta-tubulin-based assay was c. 66 times less sensitive, and therefore less suitable for detection purposes. The TaqMan-ITS assay recognized all C. acutatum isolates tested from various intraspecific molecular groups, while no amplification was observed with several other Colletotrichum species or other strawberry pathogens, indicating the specificity of this assay. Detection and quantification of C. acutatum was demonstrated in artificially and naturally infected strawberry leaves. First, C. acutatum was detected in plant mixes of which only 0.001% of the tissue was infected by C. acutatum. Secondly, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allowed monitoring of growth progression of C. acutatum. This real-time PCR-mediated monitoring of the pathogen was well-correlated with microscopic data, and confirmed that leaf age may play a role in the extent of C. acutatum infection. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material.