BackgroundAttention-deficit/hyperactivity disorder (ADHD) is a highly prevalent neurodevelopmental disorder with significant sex differences in diagnostic rates; males being diagnosed more frequently than females. Gut microbiota alterations may contribute to ADHD via the microbiota-gut-brain axis. However, previous microbiota studies have predominantly used male or mixed-gender samples, neglecting females with distinct clinical and hormonal profiles. The pre-pubertal period is critical for both neurodevelopment and microbiota maturation. This study investigates gut microbiota characteristics in pre-pubertal females with ADHD to identify sex-specific biomarkers and candidate targets for future investigation.Aim of the studyThis study aimed to characterize gut microbiota profiles in pre-pubertal females with ADHD using 16S rRNA sequencing, and to identify differentially abundant taxa and potential biomarkers through LEfSe and MetagenomeSeq analyses.Methods and materialsPre-pubertal female ADHD patients (DSM-5 diagnosed, drug-naïve) were recruited from Quzhou Third Hospital. Fecal DNA was extracted, the 16S rRNA gene amplified by PCR, and libraries sequenced on the Revio platform. Bioinformatics analysis included diversity assessment and differential abundance analysis using MetagenomeSeq and LEfSe methods.ResultsAlpha-diversity analysis revealed reduced community evenness (lower Shannon and Simpson indices) but preserved species richness (comparable Chao1) in the ADHD group. Beta-diversity analysis showed significant group differences for both Weighted and Unweighted UniFrac (PERMANOVA, P < 0.01), with a larger effect size for Unweighted UniFrac, suggesting that rare taxa may substantially contribute to community alterations. MetagenomeSeq identified Verrucomicrobiota as the only significantly elevated phylum in ADHD. LEfSe distinguished 26 differential taxa, with cross-validation designating Ruminococcus (enriched in ADHD) and Coprobacillus (depleted in ADHD) as candidate biomarkers.ConclusionPre-pubertal females with ADHD exhibited reduced gut bacterial evenness with preserved richness, characterized by higher Ruminococcus and lower Coprobacillus, suggesting lineage-specific reshuffling rather than wholesale species loss. These findings implicate gut microbiota community alterations in ADHD pathogenesis via the microbiota-gut-brain axis.