The evidence available in the pediatric population is limited for making clinical decisions regarding the optimization of tacrolimus (TAC) in pharmacotherapy. The objective of this study was to estimate the frequency of CYP3A5 genetic polymorphisms and their relationship with tacrolimus requirements in the pediatric population. This was a longitudinal cohort study with a two-year follow-up of 77 patients under 18 years old who underwent a liver transplant during the period 2009-2012 at the J.P. Garrahan Pediatric Hospital. Tacrolimus levels from day five up to two years after the transplant were obtained from hospital records of routine therapeutic drug monitoring. The genotyping of CYP3A5 (CYP3A5*1/*3 or *3/*3) was performed in liver biopsies from both the donor and the recipient. The frequency of CYP3A5*1 expression for recipients was 37.1% and 32.2% for donors. Patients who received an expresser organ showed lower Co/dose, especially following 90 days after the surgery. The role of each polymorphism is different according to the number of days after the transplant, and it must be taken into account to optimize the benefits of TAC therapy during the post-transplant induction and maintenance phases.