Machek, SB, Hwang, PS, Cardaci, TD, Wilburn, DT, Bagley, JR, Blake, DT, Galpin, AJ, and Willoughby, DS. Myosin heavy chain composition, creatine analogues, and the relationship of muscle creatine content and fast-twitch proportion to Wilks coefficient in powerlifters. J Strength Cond Res XX(X): 000–000, 2020—Little data exist on powerlifting-specific skeletal muscle adaptations, and none elucidate sex differences in powerlifters. Powerlifters tend to display higher fast-twitch fiber content and phosphagen system dependence. Nevertheless, it is unknown whether fast-twitch fiber or muscle creatine content are predictive of competitive powerlifting performance (via Wilks coefficient). Twelve actively competing powerlifters (PL; n = 6M/6F; age = 21.3 ± 1.0; 3.0 ± 1.8 year competing; 7.3 ± 6.6 meets attended) and 10 sedentary controls (CON; n = 5M/5F; age = 19.4 ± 2.0 year) underwent vastus lateralis muscle biopsies and venipuncture to compare the myosin heavy chain (MHC) fiber type and creatine analogue profiles between groups of both sexes, and determine whether MHC IIa and muscle total creatine (MTC) composition predict powerlifting performance. Samples were analyzed for specific MHC isoform (I, IIa, and IIx) content via mixed homogenate SDS-PAGE, and creatine analogues (MTC, muscle creatine transporter [SLC6A8], serum total creatine [STC], and serum creatinine [CRT]). Furthermore, MHC IIa and MTC content were compared with Wilks coefficient using Pearson correlation coefficients. Male PL MHC content was 50 ± 6% I, 45 ± 6% IIa, and 5 ± 11% IIx, versus 46 ± 6% I, 53 ± 6 IIa, and 0% IIx in female PL. Conversely, male CON MHC content was 33 ± 5% I, 38 ± 7% IIa, and 30 ± 8% IIx, vs. 35 ± 9% I, 44 ± 8% IIa, and 21 ± 17% IIx in female CON. Muscle total creatine, SLC6A8, STC, and CRT did not significantly differ between groups nor sexes. Finally, neither MHC IIa content (r = −0.288; p = 0.364) nor MTC (r = 0.488; p = 0.108) significantly predicted Wilks coefficient, suggesting these characteristics alone do not determine powerlifting skill variation.