Phylogenies make it possible to infer modes of speciation within Melampodium. This chapter begins with an analysis of isolating mechanisms in the genus, with emphasis on spatial and ecological differentiation. Phenological differences among species are rare. Another mode of isolation derives from the presence of different chromosome numbers that mirror genetic incompatibility between species. Indeed, attempts to cross taxa between different chromosomal lineages have failed, and locally co-occurring species of Melampodium, which is a common occurrence on roadsides, do possess different chromosome numbers. Detailed hybridization studies have been completed among the diploid M. gracile (n = 9) and M. microcephalum (n = 9) from Mexico, and the related tetraploid M. paniculatum (n = 18) common in Central America. Analyses of meiotic chromosome behavior in F1 hybrids have revealed that M. microcephalum was one of the diploid parents of M. paniculatum, and M. gracile was identified as the likely other parent in phylogenetic analyses.