This study investigated the psychometric properties of the Highly Sensitive Child-Rating System (HSC-RS), the existence of sensitivity groups, and the characterization of sensitivity at behavioral, genetic, and physiological levels in 541 preschoolers (M(SD)age = 3.56(0.27); 45%male; 87%Caucasian). Temperament, genetic, cortisol, and electroencephalography asymmetry data were collected in subsamples (n = 94-476). Results showed a reliable observational measure of sensitivity. Confirmatory factor and latent class analysis supported a one-factor solution and three sensitivity groups, that are a low (23.3%), medium (54.2%), and a high (22.5%) sensitivity group. Hierarchical regression analyses showed moderate associations between HSC-RS and observed temperament traits (i.e., behavioral level). In addition, a small negative association between HSC-RS and a genome-wide association study polygenic risk score (GWAS PGS) for Attention Deficit Hyperactivity Disorder was found. No relations with candidate genes, other GWAS PGS phenotypes, and physiological measures were found. Implications of our findings and possible explanations for a lack of these associations are discussed.