Abstract Summary FlexiDot is a cross-platform dotplot suite generating high quality self, pairwise and all-against-all visualizations. To improve dotplot suitability for comparison of consensus and error-prone sequences, FlexiDot harbors routines for strict and relaxed handling of ambiguities and substitutions. Our shading modules facilitate dotplot interpretation and motif identification by adding information on sequence annotations and sequence similarities. Combined with collage-like outputs, FlexiDot supports simultaneous visual screening of large sequence sets, enabling dotplot use for routine analyses. Availability and implementation FlexiDot is implemented in Python 2.7. Software and documentation are freely available at http://github.com/molbio-dresden/flexidot. Supplementary information Supplementary data are available at Bioinformatics online.