Background: Load and joint kinematics change with differences in running surface. Running regularly on trails compared to roads might influence the load on the Achilles tendon and its adaptations, along with other factors such as balance, strength, and proprioception. Objective: To investigate Achilles tendon structure and functional tests in road and trail runners. Design: Cross-sectional study. Setting: Laboratory, sport sciences college. Participants: The study included 26 road and 17 trail runners who run at least three times per week with a minimum of 20 km per week and who participated in running competitions over the preceding 2 years. Methods: Each participant was examined for Achilles tendon structure (via ultrasound tissue characterization [UTC] imaging) and underwent functional tests in addition to completing a demographic questionnaire. Main Outcome Measurements: The percentages of echo types I, II, III, and IV (degree of structural homogeneity) within the tendon, tendon length and width, tendon cross-sectional area (via UTC imaging); Ankle inversion movement discrimination ability (via Active Movement Extent Discrimination Apparatus device); dynamic postural balance (via Y balance test); jumping performance (by Triple hop distance test); and Hip muscle abduction muscle strength (by hand-held dynamometry). Results: Percentage of echo type I was significantly lower while echo type II was higher in the road group compared with the trail group (67.3% type I and 28.9% type II in the road group compared with 74.1% type I and 22.1% type II in the trail group, P <.001). No differences between genders were found and no significant differences between groups were found for the other tests. Conclusion: Tendon integrity as examined with UTC is different between road and trail runners. This suggests an influence of running surface on Achilles tendon structure. This difference was not reflected in other tests, thus the influence of tendon structure on function needs further examination.