The pantropical orchid genus Polystachya comprises approximately 230 species and shows variation in ploidy. For ongoing phylogenetic investigations in this genus, a knowledge of ploidy is necessary. We also looked for a correlation between genome size and guard cell length, so that we could estimate ploidy from preserved material and plants containing high levels of mucilage that are difficult to analyse by flow cytometry (FC). For estimates of ploidy, we measured genome size using FC. For investigations of guard cell lengths and densities, tangential cuts and nail varnish impressions were made. Diploid, tetraploid and hexaploid individuals with genome sizes ranging from 0.58 to 1.80 pg (1C-values) were found. Guard cell length ranged from 15 to 39 µm. The minimum guard cell length was greater in polyploids, but diploid species had a large range of guard cell lengths that encompassed the size range found in polyploids. Although previous studies have found that guard cell length can be used to estimate ploidy in cases in which the genome size range is large, this is not applicable to genera with a small genome size, such as Polystachya. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 223–233.