Understanding the flexibility of monocot genomes requires a phylogenetic framework, which so far is available for few of the ca. 2800 genera. Here we use a molecular tree for the South American genus Alstroemeria to place karyological information, including fluorescent in situ hybridization (FISH) signals, in an explicit evolutionary context. From a phylogeny based on plastid, nuclear, and mitochondrial sequences for most species of Alstroemeria, we selected early-branching (Chilean) and derived (Brazilian) species for which we obtained 18S-25S and 5S rDNA FISH signals; we also analyzed chromosome numbers, 1C-values, and telomere FISH signals (in two species). Chromosome counts for Alstroemeria cf. rupestris and A. pulchella confirm 2n = 16 as typical of the genus, which now has chromosomes counted for 29 of its 78 species. The rDNA sites are polymorphic both among and within species, and interstitial telomeric sites in Alstroemeria cf. rupestris suggest chromosome fusion. In spite of a constant chromosome number, closely related species of Alstroemeria differ drastically in their rDNA, indicating rapid increase, decrease, or translocations of these genes. Previously proposed Brazilian and Chilean karyotype groups are not natural, and the n = 8 chromosomes in Alstroemeria compared to n = 9 in its sister genus Bomarea may result from a Robertsonian fusion.