Background: Procedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints. Results: ProbeMaker is a software framework for computer-assisted design and analysis of sets of oligonucleotide probe sequences. The tool assists in the design of probes for sets of target sequences, incorporating sequence motifs for purposes such as amplification, visualization, or identification. An extension system allows the framework to be equipped with application-specific components for evaluation of probe sequences, and provides the possibility to include support for importing sequence data from a variety of file formats. Conclusion: ProbeMaker is a suitable tool for many different oligonucleotide design and analysis tasks, including the design of probe sets for various types of parallel genetic analyses, experim...