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Medicago lupulina L. possesses a complex of valuable economic and biological traits: high adaptive ability to climatic and edaphic growing conditions, good nutrition and digestibility of green mass. The aim of this work was to study the chloroplast genome, which is associated with plant defense responses of M. lupulina L. var. vulgaris Koch, cultivar-population VIK32, line MlS-1, which is highly responsive to mycorrhization and forms a highly effective symbiosis with salt-sensitive strain Sinorhizobium meliloti L6-AK89. The studied complete chloroplast genome is a double-stranded circular DNA molecule of 118340 bp in length with an average GC composition of 34.18%. Short inverted repeats (IRa and IRb), presumably involved in chloroplast replication processes, are 1.8 times shorter in M. lupulina VIK32 than in M. lupulinaNC_042847. Two extended regions 12501 and 5001 bp in length, presumably introduced due to HGT, and the 7 regions containing short sequences showed similarity to sequences of 260 viruses (Identity 78–96%, E-value 5 × 10–33–4 × 10–3) were identified. In addition, a number of deletions, insertions, and SNPs are significantly differentiate the chloroplast genomes at the interspecies level, this suggestion requires further investigation. The cpDNA of the M. lupulina VIK32 was shown to be phylogenetically closely related to cpDNA of M. lupulinaNC_042847 and M. minima, but it was the most divergent from the putative common Medicago-Melilotus-Glycine ancestral cpDNA sequence. [ABSTRACT FROM AUTHOR]