ABSTRACT The phylogeny of the southeastern United States species of Hymenocallis was investigated using 23 ISSR presence/absence characters and 10 qualitative and quantitative morphological characters. Maximum Parsimony (MP) and Neighbor Joining (NJ) analyses with bootstrap replicates were performed in PAUP. The total combined data NJ analysis showed several supported clades with morphological synapomorphies. Hymenocallis choctawensis was sister to H. franklinensis [83% bootstrap (bt) support]; these taxa share the morphological characters of oblanceolate leaves, few ovules and the presence of rhizomes. Hymenocallis coronaria was sister to H. liriosme (63% bt) and these taxa share the morphological feature of a prominent yellow ‘‘eye’’ in the middle of their staminal cups and both reproduce asexually by offsets rather than by rhizomes. Hymenocallis godfreyi + H. rotata + H. tridentata formed a clade (99% bt) with the morphological synapomorphy of a 2n 5 48 chromosome number. Hymenocallis henryae was sister to H. palmeri (82% bt) with several morphological synapomorphies including subcircular scapes, hyaline margins on their leaves, scape bracts that do not enclose the flowers in bud, and tepals that are colored green. Finally, H. puntagordensis and H. latifolia were sister taxa (84% bt) with the synapomorphy of persistent (versus deciduous) leaves and free filaments that are green distally. Rhizomes appear to have evolved twice within the southeastern United States species of Hymenocallis: once in the H. franklinensis + H. choctawensis + H. gholsonii + H. pygmaea + H. crassifolia + H. duvalensis clade and once in the clade containing H. godfreyi, H. rotata and H. tridentata. The alliances determined by Traub for the southeastern United States species of Hymenocallis are generally supported based on this molecular and morphological analysis.