The most important aim in interpreting an oblique ionogram is to obtain the accurate Junction Frequencies (JFs) of the ordinary (O) and extraordinary(X) mode. This requires the correct identification of O- and X-mode traces, so it is very helpful and worthy to grasp the relative position between the two modes. In this paper, a statistical and simulation study of the separation in JFs between O- and X-wave is carried out based on observed oblique ionograms over three mid-latitude paths within China and a 3-D ray tracing program. The dependences on local time, season, geomagnetic activity, solar activity, direction and length of propagation, O-wave JF and group path are investigated. The main conclusions are as follows : (a) the separation on east-west path is susceptible on ionospheric variability, while the separation on north-south path does not show a significant correlation with local time and season;(b) a general diurnal tendency and a summer anomaly on east-west propagation are first proposed and discussed, the latter may be related to the effect of the lower layers; (c)the separation varies approximately as the cosine function of the propagation direction owning two maximums at north-south direction and two minimums at east-west direction in mid-latitude region of China; (d) the variation patterns of the separation with the propagation length are obviously not the same in different directions. For the east-west propagation path, the separation decreases to a minimum near ground range of 2000km and then increases very slowly with increasing ground range, while it monotonically increases for north-south propagation path.