Accurate identification of Alternaria spp. is dependent upon the production of diagnostic morphological characters under defined cultural conditions and the proper assessment of character variation. This process is often compromised by variation in laboratory facilities and technical expertise. To assist taxon identification and phylogenetic studies, restriction site information from the intergenic spacer (IGS) region of nuclear rDNA was evaluated. Restriction maps were constructed from 15 species of Alternaria and Stemphylium botryosum (telemorph Pleospora herbarum) for 11 restriction enzymes using a new method for restriction mapping based on differential priming of IGS amplicons. IGS fragment size varied among species from 2.2–3.9 kb. Based upon restriction site homology among closely-related and more distantly related species, the IGS region could be divided into conserved and variable domains. The conserved domain was approximately 0.75 kb in size and was located at the 3′ end of the IGS region. Restriction site homology within this region was very high, especially among closely related taxa. The remainder of the region comprised the variable domain, which encompassed considerable differences in size and restriction sites among taxa. The presence or absence of restriction sites among taxa was analyzed using methods of neighbor-joining. Phylogenetic relationships based on this method were concordant with those previously resolved based upon other methods and other genomic regions.