The expression of sign-tracking (ST) phenotype over goal-tracking (GT) phenotype has been associated to different aspects of impulsive behavior, and depletions of brain serotonin (5-HT) have been shown to selectively increase impulsive action as well as ST. The present study aimed at testing the relationship between reduced brain 5-HT availability and expression of ST phenotype in a genetic model of individual variation in brain 5-HT functionality. Inbred DBA/2J (DBA) mice are homozygous for the allelic variant of the TPH-2 gene causing lower brain 5-HT function in comparison with C57BL/6J (C57) inbred mice. Young adult (10 weeks) and adult (14 weeks) C57 and DBA mice were trained in a Pavlovian conditioned approach (PCA) paradigm. Lever-directed (ST) and magazine-directed (GT) responses were measured in 12 daily conditioning sessions. In a second experiment, effect of the medial prefrontal cortex (mPFC) 5-HT depletion by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) was assessed on acquisition of ST phenotype in adult C57 mice, according to their higher 5-HT functionality compared to DBA mice. Young adult mice of both strains developed ST phenotype, but only adult DBA mice developed ST phenotype. 5-HT depletion in the mPFC of adult C57 mice completely changed their phenotype, as shown by their increased ST. These findings indicate that ST phenotype can be the expression of a transitory late developmental stage and that genetic factors determine persistence of this phenotype in adulthood. These findings also support a role of 5-HT transmission in PFC in constraining development of ST phenotype.