Aims: The purpose of this study was to determine the variability in anatoxin-a (ATX) and homoanatoxin-a (HTX) concentrations in benthic cyanobacterial mats within sampling sites and to assess the applicability of using a PCR-based approach to determine ATX- and HTX-production potential. Methods and Results: ATX and HTX variability was investigated by collecting 15 samples from 10 x 10 m grids in seven rivers. ATX and HTX concentrations were determined using liquid chromatography-mass spectrometry (LC-MS). Samples from two sites contained no ATX or HTX and at one site ATX and HTX were detected in all samples. At four sites, both toxic and nontoxic samples co-occurred and these samples were sometimes spaced less than 1 m apart. PCR amplification of a region of a polyketide synthase (ks2, putatively involved in the biosynthetic pathway of ATX and HTX) successfully distinguished ATX-and-HTX- and non-ATX-and-HTX-producing cultured Phormidium strains. Results from environmental samples were more variable, and the results were in congruence with the LC-MS data in only 58% of samples. Conclusions: Fine-scale spatial variability in ATX and HTX concentrations occurs among benthic cyanobacterial mats. Significance and Impact of the Study: Multiple benthic cyanobacterial mat samples must be collected at a sampling site to provide an accurate assessment of ATX and HTX concentrations at that location. The PCR-based technique offers the potential to be a useful early warning technique.