BACKGROUND: Micro-and minisatellites are among the most powerful genetic markers known to date. They have been used as tools for a large number of applications ranging from gene mapping to phylogenetic studies and isolate typing. However, identifying micro-and minisatellite markers on large sequence data sets is often a laborious process. RESULTS: FONZIE was designed to successively 1) perform a search for markers via the external software Tandem Repeat Finder, 2) exclude user-defined specific genomic regions, 3) screen for the size and the percent matches of each relevant marker found by Tandem Repeat Finder, 4) evaluate marker specificity (i.e., occurrence of the marker as a single copy in the genome) using BLAST2.0, 5) design minisatellite primer pairs via the external software Primer3, and 6) check the specificity of each final PCR product by BLAST. A final file returns to users all the results required to amplify markers. A biological validation of the approach was performed using the whole genome sequence of the phytopathogenic fungus Leptosphaeria maculans, showing that more than 90% of the minisatellite primer pairs generated by the pipeline amplified a PCR product, 44.8% of which showed agarose-gel resolvable polymorphism between isolates. Segregation analyses confirmed that the polymorphic minisatellites corresponded to single-locus markers. CONCLUSION: FONZIE is a stand-alone and user-friendly application developed to minimize tedious manual operations, reduce errors, and speed up the search for efficient minisatellite and microsatellite markers departing from whole-genome sequence data. This pipeline facilitates the integration of data and provides a set of specific primer sequences for PCR amplification of single-locus markers.