Any investigation of the genetic structure of populations involves the analysis of a large number of samples and therefore benefits from the use of rapid, inexpensive, and automated methods to assign individuals to a particular genotype. We developed a high throughput SNuPE (single nucleotide primer extension) assay to assess polymorphic base variations at three loci (PC-IGS, rDNA-ITS, and gvpA-IGS) in the genome of the cyanobacterium Nodularia spumigena. Using a 96-capilliary sequencer, analysis of thirteen 96-well plates can be performed in the same electrophoretic run, allowing the throughput of 1248 samples in 75 min. The SNuPE method can be broken down into two stages. The first stage comprises amplification of a DNA fragment containing the polymorphic sequence and its purification from un-incorporated PCR reagents. The second stage involves the annealing and extension of a third primer, the SNuPE primer, the 3′ end of which immediately precedes the variable site in the template. This primer is extended with a single fluorescently labeled dideoxy nucleotide by DNA polymerase, followed by characterization of the extended primers on a DNA sequencing instrument. The length of the extended primer is used to define the locus, and the incorporated fluorescent dideoxy nucleotide gives the identity of the nucleotide at each polymorphic site. Details of this technique and its application to study the genetic structure of Nodularia populations are described.