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Characterizing the influence of glacial meltwater on groundwater recharge remains a significant challenge due to the complex geology, rugged topography of high-mountain environments and overlapping isotopic fingerprints of potential recharge sources in some settings. In the Peruvian Central Andes, the Mantaro aquifer serves as the primary water source for human consumption in Huancayo city. While surface water resources are increasingly threatened by the accelerated retreat of the Huaytapallana Cordillera glaciers, the specific impact of glacial retreat on groundwater storage remains unknown. This study investigates the hydrological connectivity between Huaytapallana glacier meltwater and the Mantaro aquifer by integrating microbial DNA metabarcoding and stable isotope analysis 2H and 18O in water. We collected 32 samples including water (from glacier meltwater, lakes, springs, streams, and groundwater) and sediments in the Shullcas basin. Microbial biomass was collected by filtration and DNA was extracted. The V3-V4 region of the 16S rRNA gene was sequenced using Illumina Next-Generation Sequencing (NGS). Bioinformatics processing was conducted via the DADA2 pipeline in R platform. We analyzed microbial community composition, alpha/beta diversity, and shared taxa (ASVs/genera) to identify biological tracers. At the same time, monthly stable isotope data 2H and 18O from 2025 were analyzed using dual isotope plots to determine seasonal recharge sources.Isotopic signatures indicate that groundwater consists of a stable mixture across both dry and rainy seasons, with precipitation, glacier meltwater and surface rivers identified as primary recharge sources. Microbial analysis identified 12,989 ASVs and 695 genera. Taxonomic analysis revealed that the top 10 genera in terms of relative abundance were Hgcl clade, Flavobacterium, Brevundimonas, Sphingorhabdus, Romboutsia, Fusibacter, Pseudarthrobacter, Cypionkella, Sphingomonas, and Ferruginibacter. Alpha and beta diversity analysis and the detection of the genus CL500-29 marine group y Pseudarthrobacter in both meltwater and groundwater supports the existence of a cold, oxygenated hydrological glacial origin for recharge. Although 317 genera were shared across all sites, 12 genera were found exclusively in both glacier meltwater and groundwater. This exclusive community comprises psychrotolerant, oligotrophic, and acidotolerant taxa, including degraders of ancient organic matter (Granulicella, Cellulomonas) and taxa associated with ice environments (Subtercola, Arcticibacter).Despite the complex geology and the 20 km distance between the Huaytapallana Cordillera and the Mantaro aquifer, our findings confirm a direct hydrological and biological connectivity between the glacier and aquifer. This link is evidenced by shared microbial taxa (specifically psychrotolerant taxa) and stable isotope signatures. Currently, we are further quantifying the proportion of glacial meltwater that contributes to aquifer recharge and estimating the travel time from the glacier to the extraction wells. These ongoing analyses are crutial for predicting the long-term impact of glacier retreat on water availability. Our results emphasize the need to integrate glacial dynamics into groundwater management plans and high-mountain recharge programs to ensure sustainable water supply for the Shullcas River basin.