The patterns of distribution of a set of conserved brain developmental regulatory transcription factors and neuronal markers were analyzed in the hypothalamus of the juvenile turtle, Pseudemys scripta. Combined immunohistochemical techniques were used for the identification of the main boundaries and subdivisions in the optic, paraventricular, tuberal, and mammillary hypothalamic regions. The combination of Tbr1 and Pax6 with Nkx2.1 allowed identification of the boundary between the telencephalic preoptic area, rich in Nkx2.1 expression, and the prethalamic eminence, rich in Tbr1 expression. In addition, at this level Nkx2.2 expression defined the boundary between the telencephalon and the hypothalamus. The dorsalmost hypothalamic domain was the supraoptoparaventricular region that was defined by the expression of Otp/Pax6 and the lack of Nkx2.1/Isl1. It is subdivided into rostral, rich in Otp and Nkx2.2, and caudal, only Otp-positive, portions. Ventrally, the suprachiasmatic area was identified by its catecholaminergic groups and the lack of Otp, and could be further divided into a rostral portion, rich in Nkx2.1 and Nkx2.2, and a caudal portion, rich in Isl1 and devoid of Nkx2.1 expression. The expressions of Nkx2.1 and Isl1 defined the tuberal hypothalamus, whereas only the rostral portion expressed Otp. Its caudal boundary was evident by the lack of Isl1 in the adjacent mammillary area, which expressed Nkx2.1 and Otp. All these results provide an important set of data on the interpretation of the hypothalamic organization in a reptile, and hence make a useful contribution to the understanding of hypothalamic evolution. J. Comp. Neurol., 2012;520:453–478. © 2011 Wiley Periodicals, Inc.