The first signs of face aging appear in the midface, so procedures such as botulinum toxin and filler injections are performed there. However, no guidelines based on clinical anatomy describing the muscular and vascular components in vivo have been published. The aim of this research was to describe the depths of the midface muscles and the locations of vessels using ultrasonographic (US) imaging. US was applied at 12 landmarks on the midface in 88 volunteers (49 males and 39 females; 19-36 years) to detect sex differences in the depths of muscles and the locations of the vessels. The depths of the orbicularis oculi (OOc), levator labii superioris alaeque nasi (LLSAN), and zygomaticus minor (Zmi) differed significantly with sex at P7 (p = 0.001) and P8 (p = 0.017), P1 (p = 0.028), and P4 (p = 0.035), respectively. The facial artery, facial vein, angular artery, angular vein, and perforator vessels were found at P9, P2 and P10, P1, P1 and P5, and P8, P11 and P12, respectively. The findings indicate that the depths of the OOc, LLSAN, and Zmi muscles differ between the sexes and that the vessels appear at specific landmarks. This information could help in developing anatomical guidelines for several procedures.