Three flax (Linum usitatissimum L.) cultivars ('Ariane', 'Argos' and 'Viking') were used to study the effect of diurnal temperature range (DIF) applied throughout the whole growth stage on the development of fiber cells. Diurnal temperature ranges were set at 5C, 10C and 15C with the same daily mean temperature and accumulated growing degree days (GDDa). All measured traits showed obvious DIF and varietal differences except the length of fiber bundle (LFB) and the inter-fiber bundle distance (IFBD). Significant interactions of DIF and cultivar were observed to affect the width of fiber bundle (WFB), the size of fiber cell (SFC), the size of fiber cell cavity (SFCC) and the thickness of fiber cell wall (TFCW). Compared with other DIFs, the number of fiber cells per bundle (NFCB) and WFB increased, but IFBD decreased under DIF 5. However, WFB, TFCW and NFCB all decreased under DIF 10. Under DIF 15 condition, both SFC and SFCC increased. For 'Ariane', IFBD was short under DIF 5, but SFC and SFCC were both large under DIF 10. For 'Argos', IFBD was also short under DIF 5. Under DIF 10, WFB, SFC and TFCW were all small, but IFBD was long. For 'Viking', TFCW was small under DIF 10 but large under DIF 15. All these results suggested a definite influence of DIF on the development of fiber cells in flax, and DIF set at 5°C benefited the number of fiber cells in a bundle but DIF 15 was optimal for the size of fiber cells.