ABSTRACT: We used 18S rRNA gene metabarcoding to investigate picoeukaryotic diversity and distribution at the surface and deep chlorophyll maximum (DCM) of 4 stations in the eastern equatorial Indian Ocean (EEIO). The results showed that picoeukaryotic communities were dominated by 5 phyla: Dinoflagellata, Radiolaria, Chlorophyta, Ochrophyta and Ciliophora. The picoeukaryotic communities were classified into 3 groups matching their water mass origins and depth: (1) Group I corresponding to the surface water with Bay of Bengal extension of lower salinity, was dominated by Radiolaria Group A, Spirotrichea and Marine Stramenopiles; (2) Group II were for the DCM within the intrusion of Arabian Sea High Salinity Water, with Chloropicophyceae and Pelagophyceae being more abundant; and (3) Group III from the 0°–5°S surface water was enriched by Dinophyceae. In addition, Caecitellaceae paraparvulus was abundant at 4°S, where weak vertical mixing occurred. This study provides the first baseline of picoeukaryotic diversity in the EEIO.