The microcystin content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes and hydro-terrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys; Wrights, Victoria, Miers and Marshall. Enzyme-linked immunosorbent assays (ELISA), liquid chromatography mass spectrometry (LC-MS), and protein phosphatase inhibition assays (PP-2A) resulted in the identification of low levels (1 - 16 mg/kg dry weight) of microcystins in all samples. A plot of indicative potencies of microcystins (ratio PP-2A:ELISA) versus total microcystins (ELISA) showed a general decrease in potency as total microcystin levels increased and a clustering of values from discrete geographic locations. LC-MS/MS analysis on selected samples identified eight novel microcystin congeners. The low energy collisional activation spectra were consistent with variants of [D-Asp3] MC-RR and [D-Asp3] MC-LR containing glycine [Gly1] rather than alanine and combinations of homoarginine [hAr2] or acetyldemethyl ADDA [ADMAdda5] substitutions. Nostoc sp. was identified as a microcystin producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase (AMT) domain of the mcyE gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general samples from the same geographic location tended to clustered together. ARISA also enabled the putative identification of the microcystin producing Nostoc sp. from multiple samples.