This study evaluated the effects of different temperatures on the histological process of sex differentiation in the pejerrey Odontesthes bonariensis, a fish with marked temperature-dependent sex determination (TSD), at feminizing, neutral, and masculinizing temperatures. Fish reared at three temperatures (17°C, 24°C, and 29°C) from hatching were sampled weekly until 11 weeks and their gonads were examined by histology. The percentages of females at 17°C, 24°C, and 29°C were 100%, 73%, and 0%, respectively. Sex differentiation occurred earlier and at a smaller body size at higher temperatures in both sexes. The first signs of ovarian differentiation were observed at 4 and 7 weeks at 24°C and 17°C, respectively, and those of testicular differentiation at 4 and 7 weeks at 29°C and 24°C, respectively. Body or gonadal growth rates before sex differentiation were not proportional to temperature and showed no sexual dimorphism at 24°C, where both sexes were present. Thus, differential growth rate is probably not a factor in TSD or histological sex differentiation in pejerrey. Blood vessels were formed before sex differentiation in both sexes and at all temperatures, and may be important for sex differentiation. No signs of intersexuality were found in any of the groups, and this characterizes pejerrey as the differentiated type of gonochorist even at feminizing and masculinizing temperatures. Ovaries were formed by the same histological processes at feminizing (17°C) and neutral (24°C) temperatures and without any pathological features such as germ cell degeneration. The process of testicular formation was generally similar at 24°C and 29°C, but some fish at 29°C had widespread germ cell degeneration before sex differentiation. This suggests that pathological processes leading to germ cell death, such as heat-induced dysfunction of the supporting somatic cells, could be involved in masculinization of the genetic females at high temperatures. J. Exp. Zool. 303A:504–514, 2005. © 2005 Wiley-Liss, Inc.