Kidney transplantation (KTx) requires immunosuppressive drugs such as Tacrolimus (TAC) which is mainly metabolized by CYP3A5. TAC is routinely monitored by trough levels (C ) although it has not shown to be a reliable marker. The area-under-curve (AUC) is a more realistic measure of drug exposure, but sampling is challenging in pediatric patients. Limited-sampling strategies (LSS) have been developed to estimate AUC. Herein, we aimed to determine AUC and genotype in Chilean pediatric kidney recipients using extended-release TAC, to evaluate different LSS-AUC formulas and dose requirements. We analyzed pediatric kidney recipients using different extended-release TAC brands to determine their trapezoidal AUC and genotypes (SNP rs776746). Daily TAC dose (TAC-D mg/kg) and AUC normalized by dose were compared between CYP3A5 expressors (*1/*1 and *1/*3) and non-expressors (*3/*3). We evaluated the single and combined time-points to identify the best LSS-AUC model. We compared the performance of this model with two pediatric LSS-AUC equations for clinical validation. Fifty-one pharmacokinetic profiles were obtained from kidney recipients (age 13.1 ± 2.9 years). When normalizing AUC by TAC-D significant differences were found between CYP3A5 expressors and non-expressors (1701.9 vs. 2718.1 ng*h/mL/mg/kg, < 0.05). C had a poor fit with AUC ( = 0.5011). The model which included C , C and C , showed the best performance to predict LSS-AUC ( = 0.8765) and yielded the lowest precision error (7.1% ± 6.4%) with the lowest fraction (9.8%) of deviated AUC , in comparison to other LSS equations. Estimation of LSS-AUC with 3 time-points is an advisable and clinically useful option for pediatric kidney recipients using extended-release TAC to provide better guidance of decisions if toxicity or drug inefficacy is suspected. The different genotypes associated with variable dose requirements reinforce considering genotyping before KTx. Further multi-centric studies with admixed cohorts are needed to determine the short- and long-term clinical benefits.