The purpose of this study was to determine and explore factors (age, sex, anthropometry, running and injury/pain history, tendon gross morphology, neovascularization, ankle range of motion, and ankle plantarflexor muscle endurance) related to intra-tendinous morphological alterations of the Achilles tendon in runners. An intra-tendinous morphological change was defined as collagen fiber disorganization detected by a low peak spatial frequency radius (PSFR) obtained from spatial frequency analysis (SFA) techniques in sonography. Ninety-one runners (53 males and 38 females; 37.9 ± 11.6 years) with 8.8 ± 7.3 years of running experience participated. Height, weight, and waist and hip circumferences were recorded. Participants completed a survey about running and injury/pain history and the Victorian Institute of Sport Assessment-Achilles (VISA-A) survey. Heel raise endurance and knee-to-wall composite dorsiflexion were assessed. Brightness-mode (B-mode) sonographic images were captured longitudinally and transversely on the Achilles tendon bilaterally. Sonographic images were analyzed for gross morphology (i.e., cross-sectional area [CSA]), neovascularization, and intra-tendinous morphology (i.e., PSFR) for each participant. The factors associated with altered intra-tendinous morphology of the Achilles tendon were analyzed using a generalized linear mixed model. Multivariate analyses revealed that male sex was significantly associated with a decreased PSFR. Additionally, male sex and the presence of current Achilles tendon pain were found to be significantly related to decreased PSFR using a univariate analysis. Our findings suggested that male sex and presence of current Achilles tendon pain were related to intra-tendinous morphological alterations in the Achilles tendon of runners.