Enhancement of biomechanical stalk strength is a key pathway to improve lodging resistance in modern maize (Zea mays L.) production. Four maize varieties were used to investigate the effects of planting density on stalk penetration strength as well as agronomic traits of maize stalk. The four maize varieties were differing in lodging resistance, i. e. JK 518 and JK 519 (low density-tolerance and lodging resistant), CS 1 (high density-tolerance and lodging resistant) and ND 108 (moderate density-tolerance). Five levels of density treatment were imposed, 3.00, 5.25, 7.50, 9.25, and 12.00×104 plants/hm2, respectively). The results indicated that the stalk rind penetration strength (RPS) was enhanced with growing of maize. There was difference of stalk RPS in years, and planting densities, respectively, between maize varieties differing in density-tolerance. With the rising of internodes position, stalk RPS were decreased significantly, which fitted a quadratic function curve (y = a + bx + cx2). Furthermore, the difference of stalk RPS between varieties differing in density-tolerance was present at internodes below ear, particularly at 3rd to 6th, in the tasselling–silking stag. With increasing planting densities, stalk RPS of base internodes decreased linearly significantly. Therefore, in the pre-tasselling stage, developed early and thicken in stalk wall indicates that stalk RPS was higher. In the silking stage, both shorter internodes below ear and well developed stalk base had the benefit for improving the stalk lodging resistance. In conclusion, that the 7 days before tasselling–silking stag and 3rd to 6th of the base internodes can be used as the suitable test time and sensitive internodes of the stalk of rind penetration strength in lodging resistance identification. This method can evaluate the stalk strength objectively and improve the breeding efficiency of selection varieties with lodging resistance.