Lakes cover large parts of the climatically sensitive Arctic landscape and respond rapidly to environmental change. Freshwater diatoms dominate the primary producer community in these lakes and can be used to detect biotic responses to climate and environmental change. We used specific diatom metabarcoding on sedimentary DNA, combined with next-generation sequencing and diatom morphology, to assess diatom diversity in five glacial and 15 thermokarst lakes within the easternmost expanse of the Siberian treeline ecotone in Chukotka, Russia. We obtained 163 verified diatom sequence types and identified 176 diatom species morphologically. Raw and resampled read counts of diatom DNA sequences retrieved from our approach are and its taxonomic classifications are listed in the spread sheet: Huang_16KP_diat_JOPL.xlsx. The retrieved diatom sequences are listed in the fasta file: 16KP_diat_sequences.fasta. Raw and resampled valve counts of morphologically identified diatom taxa are summarized in the spreadsheet Huang_16KP_diat_JOPL.xlsx.