A culture-independent method for multilocus sequence typing of Microcoleus chthonoplastes was developed based on mechanical separation of individual cyanobacterial filaments from natural microbial mat populations through micromanipulation, subsequent polymerase chain reaction (PCR) amplification and sequence analysis of three genetic loci (kaiC, petB/D, rDNA-ITS). Among 81 individuals sampled from intertidal sand flats of the North Sea and Baltic Sea, we found 8-14 different sequences (alleles) per genetic locus, resulting in 36 distinct genotypes with unique allele profiles. Non-congruent phylogenetic gene trees for the three loci analysed and split decomposition analysis indicated the occurrence of horizontal genetic exchange. The index of association determined for the entire population was 0.096, indicating that recombination occurs frequently enough to cause almost random association (linkage equilibrium) among alleles. Analysing individuals from three different locations in the North Sea and Baltic Sea, we did not find evidence for geographic subdivisions between populations.