The present knowledge of karyological characters, including basic numbers, ploidy levels, chromosome structures, karyotypes, interphase nuclear types, and DNA contents, and the broad range of variation for most of these characters within Rubiaceae (excluding Rubieae) are summarized. These data are related to the presently existing systematic concepts of the family. The use of karyological characters is exemplified in some taxonomically critical groups: Subfamily Antirheoideae sensu Robbrecht appears heterogeneous with regard to basic numbers and chromosome structure; it contains chromosomally homogeneous tribes (e.g., Vanguerieae or Guettardeae), but also groups with internal variation (such as the tribe Knoxieae or the genus Chiococca). Available chromosome data of the tribe Cinchoneae sensu Robbrecht seem to be in accordance with its morphologically and molecular-based subdivision in three tribes: viz. Cinchoneae s. str., Calycophylleae, and Coptosapelteae. The tribe Chiococceae sensu Bremer consists of at least two distinct entities characterized by their chromosome morphology and basic numbers: the former tribe Condamineeae, and the genera Chiococca and Exostema. A possible subdivision of Rondeletia is indicated by three different basic numbers (x = 9, x = 10, x = 11) occurring in the genus, while chromosome data give no hints regarding the position of Wendlandia and Sipanea in the tribe Rondeletieae. The tribe Isertieae comprises genera with basic numbers of x = 9, x = 10, and x = 11. Thus the tribal position of Acranthera (x = 10), Heinsia (x = 11), and Mycetia (x = 11) cannot be decided on the basis of chromosome numbers alone. Sabicea has taxa with either x = 9 or x = 11; morphological studies are required to check the generic delimitations. The close relationship of Panridiantha and Urophyllum is supported by karyomorphology and basic number. A possible origin of the Spermacoce out of the tribe Hedyotideae is discussed.