Setiechinopsis is a monotypic Argentine endemic genus (S. mirabilis) of the tribe Trichocereeae. It is one of the most difficult cacti to find and grows in lowlands and brackish soils. Our aim was to analyze for the first time its cytogenetic features in two populations. It presented 2n = 22 with small chromosomes (mean chromosome length = 2.87 μm; mean haploid genome length = 31.60 μm). The karyotype was symmetrical: 10m 1sm. The first m pair (No. 1) had nucleolar organizing regions and terminal microsatellites on the short arms. The ratio of the length of the largest m chromosome pair (No. 1) to that of the smallest m chromosome pair (No. 10) was 1.55. Banding patterns showed CMA /DAPI− constitutive NOR-associated heterochromatin in m chromosome pair No. 1, comprising the distal satellite and a small proximal band. Additionally, four m chromosome pairs showed CMA /DAPI− pericentromeric bands. The percentage of CMA /DAPI− heterochromatin was 11.22% of the total karyotype length. No CMA−/DAPI bands were detected. The signal of the 18-5.8-26S gene was located in the satellite and the terminal portion of the short arm of pair No. 1. The signals of the 5S rDNA gene were located in pericentromeric regions in m chromosome pairs Nos. 2–5. The locations of the 18-5.8-26S sites coincided with the NOR-associated CMA /DAPI− bands, whereas 5S sites coincided with the pericentromeric CMA /DAPI− bands. Sizes, numbers, and intensities of both rDNA signals had a great similarity between the homologs. Comparisons with the few studies made in the Cactaceae suggest that morphological variation in the family was not followed by major modifications in karyotype formulae and chromosome size, but that the occurrence and distribution of different repetitive DNA fragments tend to vary among the different taxa so far analyzed.