Genetic mapping and the selection of closely linked molecular markers for important agronomic traits require efficient, large-scale genotyping methods. A semi-automated multifluorophore technique was applied for genotyping AFLP marker loci in barley and wheat. In comparison to conventional P-33-based AFLP analysis the technique showed a higher resolution of amplicons, thus increasing the number of distinguishable fragments. Automated sizing of the same fragment in different lanes or different gels showed high conformity, allowing subsequent unambigous allele-typing. Simultaneous electrophoresis of different AFLP samples in one lane (multi-mixing), as well as simultaneous amplification of AFLP fragments with different primer combinations in one reaction (multiplexing), displayed consistent results with respect to fragment number, polymorphic peaks and correct size-calling. The accuracy of semi-automated codominant analysis for hemizygous AFLP markers in an F-2 population was too low, proposing the use of dominant allele-typing defaults. Nevertheless, the efficiency of genetic mapping, especially of complex plant genomes, will be accelerated by combining the presented genotyping procedures.