Dysregulated fear conditioned responses have been associated with PTSD in adults, with increased fear-potentiated startle (FPS) serving as a potential intermediate phenotype for PTSD risk. This phenotype has also been associated with stress-related ADCYAP1R1 gene variants in adult women. However, FPS and genotype have not yet been examined during development. The aim of this study was to examine developmental changes in fear conditioning, and to see whether these changes were impacted by genotype and trauma. Differential fear conditioning using FPS was tested in n = 63 children ages 8-13 at two visits (V1, V2) 1 year apart. Startle response was measured using electromyograph recordings of the eyeblink muscle. The rs2267735 SNP of the ADCYAP1R1 gene was extracted from genome-wide (GWAS) analyses. Trauma exposure was assessed using the Violence Exposure Scale-Revised (VEX-R). We found significant Visit by Genotype interactions, with CC genotype increasing FPS from V1 to V2. At V2 there was a Genotype by Violence interaction, with higher FPS in the CC vs G allele groups among those with higher violence exposure (F = 17.46, p = 0.0002). Females with the CC genotype had higher FPS compared to G allele females (F = 12.09, p = 0.002); there were no effects of genotype in males. This study showed Gene × Environment × Development and Gene × Sex effects of ADCYAP1R1 in a high-risk pediatric population. Those with the CC genotype and high levels of violence exposure, as well as females with the CC genotype, showed the greatest conditioned fear responses in adolescence.