Objectives: This study aims to evaluate the effects of anchor positions on the suture holding strength of a double-row knotless fixation in rotator cuff repair. Materials and methods: Four different double-row fixation techniques were assessed. In group 1, a 15-mm-wide mattress suture was fixed using a knotless lateral row anchor, horizontal to the shaft. In group 2, the medial sutures were fixed with a 5-mm more lateral anchor that was placed at 45° to the long axis of the humeral shaft. In group 3, different from group 2, medial sutures were fixed with a 30-mm mattress suture width. In group 4, the mattress sutures coming from the medial row anchors were fixed to the 10-mm more lateral row, vertical to the long axis of the humeral shaft. The specimens were cyclically loaded from 10 N to 30 N at 0.5 Hz for 50 cycles, and then loaded to failure. Results: Group 4 had higher cyclic elongation values than group 1 (p=0.021) and group 3 (p=0.006). Group 1 had lower maximum load value than group 3 (p=0.011). Most of the specimens failed with suture ruptures. Unlike the other groups, none of the specimens in group 4 failed via a suture pull through the lateral anchor. Conclusion: A horizontal lateral row anchor positioned closer to the medial anchor resulted in less cyclic elongation when compared to a more vertically positioned lateral row anchor. The vertical or oblique positioning of the lateral row anchor did not result in any increase in the failure load value; however, the vertical placement prevented a suture pull through the lateral row anchor.