Four sterile lines (Peiai64S, Y58S, Guangzhan63–2S and H638S) and the restorer line R527 were used as materials. Five temperature gradients(24, 27, 30, 33 and 36 ℃ in artificial climate chamber) and the natural temperature (as control)were treated to the four sterile lines for 6 days at the fertile sensitive stage of heading to flowering stage respectively, to study the effect of temperature on physiological biochemical characteristics of young spike and outcrossing characteristics. The results showed that the stigma exposure rates of Peiai64S and Y58S were highest at 27 ℃, which were 86.81% and 86.06% respectively, while the stigma exposure rates of Guangzhan63–2S and H638S were highest at 24 ℃, they were 76.24% and 81.76%; the stigma vitalities of Peiai64S, Y58S, Guangzhan63–2S, and H638S were best at 24 ℃, which were 1.96, 2.12, 1.74 and 1.94, respectively; the outcrossing rates of Peiai64S, Guangzhan63–2S and H638S were highest at 24 ℃, which were 58.87%, 54.22% and 50.50% respectively, while the outcrossing rate of Y58S was highest at 27 ℃, which was 58.96%; the content of peroxidase (POD) of four sterile lines increased significantly at 33 ℃ compared with the control, while the content of malondialdehyde (MDA) and proline increased significantly at 36 ℃ compared with the control. There were differences in temperature sensitivity between the male sterile lines, 24 ℃ treatment during the sensitive period was the best for Peiai64S fertility–sensitive period, while 27 ℃ was the best temperature for Y58S, Gangzhan63–2S, H638S.