Alternaria alternata, A. radicina and A. dauci are important seed-borne fungi on carrot, with the first two species having a high toxigenic potential, for which a specific and sensitive detection method is required. Because both the traditional deep-freeze-blotter method and plating on selective medium are time consuming and laborious, a PCR-based assay was developed. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) from 45 different Alternaria isolates were determined, a restriction fragment length polymorphism analysis was performed and a phylogenetic tree was constructed. Based on the sequences, specific primers for detection and identification of the three Alternaria species on carrot seeds and roots were designed. The primers were highly sensitive and were shown to be able to differentiate between the three Alternaria species. A. alternata and A. radicina could be detected in DNA isolated from carrot material applying the specific primers, even at low infection levels. The PCR-assay was compared to the deep-freeze-blotter method (DFBM) and plating on Alternaria radicina Selective Agar (ARSA, for A. radicina) by testing naturally infected seed samples and root material. Results of the PCR-assay were similar to those of the blotter method and plating on ARSA for the detection of A. alternata and A. radicina. A positive correlation was found between the percentage of seed infection established by the blotter method and the intensity of the amplified, specific product. The PCR-assay based on the specific primers developed seems to be a good alternative for the deep-freeze-blotter method and plating on ARSA, especially when time is an important issue.