The New World endemic genus Zeltnera consists of 25 species mainly distributed in the western part of the United States and Mexico. Chromosome counts performed on 113 populations (24 species) reveal extensive congruence between chromosomal groups and the assemblages obtained from analyses of nuclear ribosomal DNA (ITS) and chloroplast DNA (trnL intron and trnL-F intergenic spacer) sequences. Karyological and molecular data sets support three main biogeographic groups for Zeltnera. A first and mainly unresolved cluster (n = 17 and n = 20) occurs in California, whereas two other clades are centered in the Texas region (n = 20 and n = 21) and in Mexico (n = 21 and n = 22). Under the assumption of a molecular clock, and using both dispersal and vicariance explanations for the current distribution of the respective species, the genus is thought to have a North American origin with considerable diversification in the early Pliocene (ca. 5 million years ago). Geological events, such as desert formation and mountain orogenies, have created insuperable barriers that today separate the three major and likely vicariant groups.