Testate amoeba-derived transfer functions are frequently used in peatland palaeohydrological studies and involve the development of training sets from surficial peats. However, within acrotelmic peats, considerable vertical variation in assemblage composition can occur, particularly along Sphagnum stems, which may limit the representation of the associated ‘contemporary’ testate amoeba samples as analogues for the peatland surface. This paper presents contiguous testate amoeba assemblage data from nine monoliths collected from different peatland microforms (hummock, hollow, lawn) in three Sphagnum dominated ombrotrophic peatlands in Ontario and Quebec, eastern Canada. The aim is to: i) gain a greater understanding of the vertical distribution of xerophilous/hygrophilous taxa along Sphagnum stems; ii) determine the vertical extent of live/encysted taxa along this gradient; and iii) assess the significance of this distribution on surface sampling protocols. The results show that testate amoeba communities in the uppermost acrotelmic peat layers display considerable variability. This may reflect a complex interplay of abiotic and biotic controls, including moisture, temperature, light and other characteristics, food availability, and mineral particle availability for test construction. These findings underline the complexity of testate amoeba community structure and highlight the importance of analysing both living and dead Sphagnum stem sections when developing calibration sets.