The evolutionarily conserved Nkx6 family transcription factors play important roles in the patterning of the central nervous system (CNS) and pancreas in vertebrates. In this study, we describe the cloning and expression patterns of the three Nkx6 family genes in Xenopus laevis. Like their mouse and chicken homologues, Xenopus Nkx6 family genes are mainly expressed in the CNS and anterior endodermal tissues during embryonic development. Nkx6.1 and Nkx6.2 share overlapping expression domains in the ventral neural tube at neurula stages and later in the ventral part of developing hindbrain and spinal cord. Nkx6.3 is detected in the non-neural ectoderm from cleavage to early neurula stages and in the caudal hindbrain and the mandibular arch at tail bud stages. In the endoderm, Nkx6.2 is expressed in the hypochord at tail bud stages. At tadpole stages, the three Nkx6 genes are differentially expressed in the anterior endoderm derivatives, including the pancreas, stomach, esophagus, and lung.