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Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. <i>Medicago truncatula</i> is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, <i>M. truncatula</i> reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. <i>M. truncatula</i> R108 is more attractive for genetic studies due to its high transformation efficiency and <i>Tnt1</i>-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for <i>M. truncatula</i> cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the <i>Tnt1</i> insertion landscape on this reference assembly presents an important resource for <i>M. truncatula</i> functional genomics by supporting efficient mutant gene identification in <i>Tnt1</i> insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future.