As High Arctic environments are particularly sensitive to global and regional climate changes, a growing number of studies have focused on that region. It has been shown that living and fossil diatoms can be successfully used to track environmental changes in polar habitats. Nevertheless, the diatom flora of many Arctic areas remains unknown. The present study set out to examine the diatom flora in the rarely visited and near-pristine zone of northern Spitsbergen. Examination by light and scanning electron microscopy of 25 sediment samples, collected in fjords, tidal plains and lakes, indicated significant differences between the diatom assemblages identified in lakes located within different fjord watersheds. Altogether, 96 diatom taxa (46 genera) were found. The most abundant species (Achnanthidium minutissimum, Staurosirella pinnata and Nitzschia alpina) occurred in at least eight of the 11 investigated lakes. Assemblages from the Woodfjorden region were characterized by the presence of Cavinula pseudoscutiformis and Encyonema reichardtii, along with Navicula spp., which coincided with relatively low conductivity (34–58.7 µS cm−1) and near-neutral pH (7.2–7.5). Diatom assemblages found in the Wijdefjorden area were typically characterized by Denticula kuetzingii and Nitzschia inconspicua, with these lakes generally having higher water conductivity (>184 µS cm−1) and pH (7.5–8.1) conditions. Conductivity, biogenic silica concentration and water temperature were indicated as significant predictors of diatom community species composition and structure. No diatom frustules were found in fjord and tidal plain sediments. The effects of selected environmental factors on diatom assemblage formation are discussed.