| Summary: | <i>Trifolium</i> L., which belongs to the IR lacking clade (IRLC), is one of the largest genera in the Leguminosae and contains several economically important fodder species. Here, we present whole chloroplast (cp) genome sequencing and annotation of two important annual grasses, <i>Trifolium alexandrinum</i> (Egyptian clover) and <i>T. resupinatum</i> (Persian clover). Abundant single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels) were discovered between those two species. Global alignment of <i>T. alexandrinum</i> and <i>T. resupinatum</i> to a further thirteen <i>Trifolium</i> species revealed a large amount of rearrangement and repetitive events in these fifteen species. As hypothetical cp open reading frame (ORF) and RNA polymerase subunits, <i>ycf1</i> and <i>rpoC2</i> in the cp genomes both contain vast repetitive sequences and observed high Pi values (0.7008, 0.3982) between <i>T. alexandrinum</i> and <i>T. resupinatum</i>. Thus they could be considered as the candidate genes for phylogenetic analysis of <i>Trifolium</i> species. In addition, the divergence time of those IR lacking <i>Trifolium</i> species ranged from 84.8505 Mya to 4.7720 Mya. This study will provide insight into the evolution of <i>Trifolium</i> species.
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