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Color pattern is recognized as an important characteristic for diagnosing Trichomycterus species and for elucidating their relationships. An analysis based on morphological and molecular data confirms the existence of a single species of Trichomycterus in the rio Itatinga, a costal river drainage on the escarpment of the Serra do Mar and the rio Claro on the upper course of the rio Tietê. The only species found, Trichomycterus iheringi, shows two clearly distinct patterns of body pigmentation and intermediate color patterns related to body size and microhabitat preference.