Sisyrinchium micranthum is native to southern Brazil and shows a wide range of morphological variation in this region. This study was performed to compare individuals of this species in terms of traits such as morphology, chromosome number, meiotic behaviour, pollen viability and genetic diversity using inter simple sequence repeat markers. First, general morphological aspects were evaluated and plants were classified into three morphological categories (CI, CII and CIII) to analyse the cytogenetic and genetic data. Three chromosome numbers corresponding to three ploidies were found: 2n = 2x = 16, 2n = 4x = 32 and 2n = 6x = 48. All morphological types showed regular meiotic behaviour and high meiotic index values and pollen viability. CII is the most frequent in southern Brazil. The most common haploid chromosome number, n = 8, has not been reported previously in this species. The percentage of polymorphic loci ranged from 6.45 to 61.29% for each accession. Molecular and cytogenetic analyses indicate that some S. micranthum accessions may have more than one ploidy. This study represents the first effort to characterize this taxonomically complex species based on cytogenetic and genetic aspects. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 350–364.