The microstructure and crystallographic characteristics of diamond film deposited with the chemical vapor deposition method was investigated using transmission electron microscopy (TEM). It is found that fivefold twin center does not always behave as growth nuclei of diamond grain. For those behaving as growth nuclei, there are a large amount of defects in every block of fivefold twin. However, for those not behaving as growth nuclei, two blocks of fivefold twin contain large quantities of defects, the other three are nearly clear. High resolution TEM analysis shows stacking faults coexist with microtwins in the two vast-defects-contained blocks. The effect of the mismatch angle (7.5°) in fivefold twins on microtwins, and the coexistence mechanism of both stacking faults and microtwins are also discussed.