Guidelines for assessing the function of gluteus minimus and gluteus medius with electromyography (EMG) traditionally offer one electrode placement site per muscle. However, anatomical studies suggest that there are two uniquely oriented segments within gluteus minimus (anterior and posterior), and three within gluteus medius (anterior, middle, and posterior) with potential for independent function. Assessment of these muscles with one electrode may therefore provide only a limited account of their role. Thus, the aim of this cadaveric study was to verify guidelines for placing intramuscular electrodes into two uniquely oriented segments of gluteus minimus, and three segments of gluteus medius. The guidelines were developed with reference to anatomical reports, cadaveric observation and real-time ultrasound imaging in vivo. Five cadaveric gluteal regions were marked for intramuscular electrode insertions based on these guidelines. Intramuscular electrodes were inserted into the marked regions of gluteus minimus (2×) and gluteus medius (3×) with the aid of a 15 cm biopsy needle. Systematic dissection revealed that electrodes were successfully inserted into uniquely oriented segments of gluteus minimus and medius. The orientation of fascicles surrounding each electrode was also consistent with segmental descriptions in past anatomical research. The findings of this research suggest that the guidelines described may be used to assess the functional role of segments within gluteus minimus and medius in health and dysfunction using EMG. Finally, electromyographers intent on investigating the role of posterior gluteus minimus must be cautious of the superior gluteal neurovascular bundle.