Restriction fragment length polymorphism tools is an R application which supports a complete workflow of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), dealing with the problems which accompany analysis when PCR-RFLP is used in diversity studies. Large numbers of different RFLP samples obtained from multiple electrophoresis runs might lead to limitations or misidentifications due to the need for band matching in most existing software applications. Due to the common problem of variation in the density of bands (i.e. distances between bands or visual intensity) in the electropherograms, it is desirable to have options for handling samples with uncertain or faint bands. As a further step in the workflow, scientists often use DNA sequencing to identify individual genotypes, so that the use of specific software to combine these tasks might be helpful. With this background, we here present an application that supports a complete workflow, starting with the analysis of single species samples by PCR-RFLP, to PCR-RFLP genotype identification based on a reference data set and DNA sequencing followed by similarity analysis. RFLPtools is a freely available, platform-independent application which provides analysis functions for DNA fragment molecular weights (e.g. by RFLP analysis), including similarity calculations without the need for band matching. As it is written for the statistical software R, other statistical analyses might also be easily applied.