Technical Abstract: The automated generation of amplified fragment length polymorphism (AFLP) markers using capillary array systems and fluorescently labeled primers is appealing to researchers with large numbers of samples. Of the commercially available equipment for automated fragment analysis, Applied Biosystems (Foster City, CA, USA) sells several of the most widely used capillary array systems. Unfortunately, GeneMapper, the latest fragment analysis software from Applied Biosystems, does not support AFLP analysis. This paper describes how to optimize GeneMapper parameters for automated AFLP fragment sizing and introduces a novel Excel macro that converts GeneMapper output into a binary alignment compatible with phylogenetic analysis software. A semi-automated approach to AFLP using GeneMapper enables researchers with Applied Biosystems instruments to produce quality data with a minimum of manual editing and formatting.