Bacterial communities in Arctic sea ice play an important role in the regulation of nutrient and energy dynamics in the Arctic Ocean. Sea ice has vertical gradients in temperature, brine salinity and volume, and light and UV levels. Multiyear ice (MYI) has at least two distinct ice layers: old fresh ice with limited permeability, and new saline ice, and may also include a surface melt pond layer. Here, we determine whether bacterial communities: 1) differ with ice depth due to strong physical and chemical gradients, 2) are relatively homogenous within a layer, but differ between layers, or 3) do not vary with ice depth. Cores of MYI off northern Ellesmere Island, NU, Canada were sub-sectioned in 30 cm intervals and the bacterial assemblage structure was characterized using 16S rRNA gene pyrotag sequencing. Assemblages clustered into three distinct groups: top (0-30 cm); middle (30-150 cm); and bottom (150-236 cm). These layers correspond to the occurrence of refrozen melt pond ice, at least two-year old ice, and newly grown first-year ice at the bottom of the ice sheet, respectively. Thus, MYI houses multiple distinct bacterial assemblages and in-situ conditions appear to play a less important role in structuring microbial assemblages than the age or conditions of the ice at the time of formation.This article is protected by copyright. All rights reserved.