Background With about 100 species, Commelina is the largest genus of Commelinaceae in Africa. Although medicinal and economic benefits had been studied extensively, little is known about its cytological analysis. Hence, this study will focus on chromosome and nucleoli analysis of selected species of Commelina L. Somatic chromosomes were prepared from root tips that emerged from the nodes of stem cuttings that were made to stand submerged in water. The roots were pretreated in 8-hydroxyquinelin 3–5 hrs followed by fixation in 3:1 ethanol: acetic acid for 1–24 hrs at 4OC. Air-dry slides were prepared following cellulase and pectinase maceration at 37OC, the preparation was stained in Giemsa stain (PH 6.4), rinsed and mounted. Nucleoli were stained in silver nitrate solution.Results Chromosome numbers and Karyotype formula of the four species were found as C. africana 2n = 2x = 30 (12m + 10sm + 8st), C. benghalensis 2n = 6x = 66 (36m + 24sm + 6st), C. diffusa (Ginchi) 2n = 66 (28m + 26sm + 12st), C. diffusa (Jimma) 2n = 2x = 30 (10m + 8sm + 12st) and C. subulata 2n = 2x = 30 (18m + 10sm + 2st). According to Stebbins karyotype asymmetry, the karyotypes of C. africana and C. subulata were 2A type, while that of C. benghalensis and C. diffusa (Ginchi) were 2B type. 3A asymmetry type was obtained for C. diffusa (Jimma). Karotypes of Tradescantia were found to be monomodal for the Commelina species of the studied plant materials. Satellites were observed for species C. africana and C. diffusa with variation in number ranging from 2 to 6. The maximum number of nucleoli observed varies from two to four for Commelina.Conclusions This study reported karyotype and nucleoli of the Ethiopian Commelinaceae for the first time. The current investigation can be considered as an additional karyotype data to the earlier meiosis report for Ethiopian materials.