This paper presents chromosome data for 130 accessions of 91 species or subspecific taxa representing 26 genera of Neotropical Rubioideae (Rubiaceae). It includes the first chromosome counts for the tribe Perameae, for 11 genera, and for 71 species and subspecific taxa. A survey of the karyological knowledge (chromosome numbers, ploidy levels, chromosome morphology) for Neotropical Rubioideae is given. The relevance of the karyological information for a discussion on relationships and speciation in the Rubioideae is commented on. Possible taxonomic implications of the data are presented.