Attention deficit-hyperactivity disorder (ADHD) is a neuropsychiatric disorder characterized by inappropriate and impaired levels of hyperactivity, impulsivity and inattention. Around 75% of adults with ADHD show comorbidity with other psychiatric disorders such as disruptive behavior disorders or substance use disorders (SUDs). Recently, there has been growing interest in studying the role of microRNAs (miRNAs) in the susceptibility to complex disorders. Interestingly, converging evidence suggests that single nucleotide polymorphisms (SNPs) within miRNAs or miRNA target sites may modulate the miRNA-mediated regulation of gene expression through the alteration of the miRNA maturation, structure or expression pattern as well as the silencing mechanisms of target genes. Genetic studies and animal models support the involvement of the serotonin receptor (HTR1B) in ADHD. We evaluated the contribution of one SNP in the miR-96 target site at HTR1B and eight tagSNPs within the genomic region containing this miRNA in 695 adults with ADHD (266 and 396 subjects with and without comorbid SUD, respectively), 403 subjects with SUD without life-time diagnosis of ADHD and 485 sex-matched controls from Spain. Single and multiple marker analyses revealed association between two SNPs located at the 3' region of miR-96 (rs2402959 and rs6965643) and ADHD without SUD. Our results provide preliminary evidence for the contribution of two sequence variants at the miR-183-96-182 cluster to ADHD without comorbid SUD, and emphasize the need to take comorbidities into account in genetic studies to minimize the effect of heterogeneity and to clarify these complex phenotypes.