<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Microsatellites (MSs) are DNA markers with high analytical power, which are widely used in population genetics, genetic mapping, and forensic studies. Currently available software solutions for high-throughput MS design (i) have shortcomings in detecting and distinguishing imperfect and perfect MSs, (ii) lack often necessary interactive design steps, and (iii) do not allow for the development of primers for multiplex amplifications. We present a set of new tools implemented as extensions to the S<jats:sc>TADEN</jats:sc> package, which provides the backbone functionality for flexible sequence analysis workflows. The possibility to assemble overlapping reads into unique contigs (provided by the base functionality of the S<jats:sc>TADEN</jats:sc> package) is important to avoid developing redundant markers, a feature missing from most other similar tools.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Our extensions to the S<jats:sc>TADEN</jats:sc> package provide the following functionality to facilitate microsatellite (and also minisatellite) marker design: The new modules (i) integrate the state-of-the-art tandem repeat detection and analysis software P<jats:sc>HOBOS</jats:sc> into workflows, (ii) provide two separate repeat detection steps – with different search criteria – one for masking repetitive regions during assembly of sequencing reads and the other for designing repeat-flanking primers for MS candidate loci, (iii) incorporate the widely used primer design program P<jats:sc>RIMER</jats:sc> 3 into S<jats:sc>TADEN</jats:sc> workflows, enabling the interactive design and visualization of flanking primers for microsatellites, and (iv) provide the functionality to find optimal locus- and primer pair combinations for multiplex primer design. Furthermore, our extensions include a module for storing analysis results in an SQLite database, providing a transparent solution for data access from within as well as from outside of the S<jats:sc>TADEN</jats:sc> Package.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>The S<jats:sc>TADEN</jats:sc> package is enhanced by our modules into a highly flexible, high-throughput, interactive tool for conventional and multiplex microsatellite marker design. It gives the user detailed control over the workflow, enabling flexible combinations of manual and automated analysis steps. The software is available under the OpenBSD License [1, 2]. The high efficiency of our automated marker design workflow has been confirmed in three microsatellite development projects.</jats:p> </jats:sec>