There is growing interest in the development of high-amylose cereals such as wheat due to their functional properties and nutritional value in foods. The increase in amylose content is associated with significant changes in the physicochemical, molecular, and structural characteristics of wheat starch which could affect its processing quality. The present study aimed to analyze the physicochemical, molecular, and structural characteristics of high-amylose starch from three durum wheat near isogenic lines (NILs), each obtained using three different recurrent parents. Morphology, granule size distribution, pasting and thermal properties, X-ray diffraction, and structural features were determined. The NILs showed the highest amylose content (65.3 and 69.8%), granules with an elongated shape, size between the wild-type and the mutant, restricted swelling in the pasting profile, the highest average and final gelatinization temperature, and B-polymorphism. The starches from the NILs showed the highest molar mass of amylopectin (AP) (7.0–7.6 × 107 Da) and the lowest amylose (AM) (1.1–1.8 × 106 Da), an issue associated with the biosynthesis of both components. The study on the debranched sample by high-performance size-exclusion chromatography showed that the high-amylose starch had the highest content of long B2 and B3 chains of AP (23.9–26.6%), which was corroborated with the high-performance anion-exchange chromatography analysis where the chains with degree of polymerization >37 had the highest content (18.3–19.1%). The PC1 is highly associated with the MwAM and the AM fraction in the debranched starch analyzed by HPSEC. Characterizing high-amylose durum wheat starch provides information to determine the structure-function relationship and design strategies to produce crosses with structural characteristics of AM and AP for specific applications.