Abstract Context Recent genetic studies have identified genetic variants associated with age at pubertal onset. Whereas genome-wide association studies reported associations of several hundred genetic variants with timing of self-reported age at menarche, a recent clinical study focused on genetic variation affecting follicle-stimulating hormone action and clinically determined age at thelarche. The observations appear to be incongruent, as effect sizes varied substantially among the studies. Alternatively, this may point to a differential impact of specific genetic loci on distinct pubertal events. Objective To investigate whether top-candidate genetic variants exhibit a different impact on timing of thelarche vs menarche, respectively. Design Cross-sectional and longitudinal study of healthy girls. Setting Population-based study in the Copenhagen area. Patients or Other Participants Girls (1478) were followed through puberty and genotyped for FSHB c.−211G>T (rs10835638), FSHR c.−29G>A (rs1394205), FSHR c.2039A>G (rs6116), LIN28B (rs7759938), INHA (rs4141153), MKRN3 (rs12148769), TMEM38B (rs10453225), and ZNF483 (rs10980921). Main Outcome Measures Clinical pubertal staging and anthropometric data. Results We observed an association of LIN28B (rs7759938) with age at thelarche (P < 0.001, effect size: 0.27 year, 95% confidence interval: 0.12 to 0.42) and age at menarche (P = 0.005, 0.17 year, 0.05 to 0.29). FSHB c.−211G>T (rs10835638) and FSHR c.−29G>A (rs1394205) minor allele count was associated with age at thelarche (P = 0.004, 0.19 year, 0.06 to 0.31) but not with age at menarche (P = 0.97; all adjusted for body mass index z scores). Conclusion Our results indicate a differential impact of specific genetic loci on age at thelarche and menarche in healthy girls.