Leeches are significant medicinal invertebrates, particularly well-known for their strong bioactive substances and therapeutic uses in analgesia, anticoagulation, and anti-inflammatory conditions. Despite this, insufficient information is available about the molecular processes driving species-specific functional diversity. We conducted a comparative transcriptomic study of three medically valuable leech species, Hirudinaria manillensis, Hirudo nipponia, and Whitmania pigra, and explored the interspecies differences in gene expression and regulatory networks. In total, 18,107 mRNAs and 183 long non-coding RNAs (lncRNAs) were identified from 27 cDNA libraries, and pairwise comparison differential gene expression analysis retrieved 1681, 2156, and 468 DEGs in HM vs HN, HM vs WP, and HN vs WP, respectively, with 81 common genes among all species. These DEGs are mainly enriched in muscle growth, metabolic regulation, and cytoskeletal structure. Muscle-related genes (TPM, TNNT, and TNNI) exhibited higher expressions in H. nipponia, suggesting greater contractile adaptability, whereas H. manillensis displayed high expression of anticoagulant (HIRM2, ANTA, FLNA, PROS), analgesic (AGRIN, MMEL1, NEP), and anti-inflammatory (MMP17, MMP25, TLR4, CYTB) genes, highlighting its robust pharmacological potential. In contrast, W. pigra had comparatively lower expression of these functional genes. Besides, co-expression evaluation presented DElncRNAs, including MSTRG.1617, MSTRG.484, and MSTRG.1673 were shown to be crucial cis-regulators of muscle and bioactive genes (TPM, TNNT, PROS, and MMP25), demonstrating intricate lncRNA–mRNA connections influencing physiological specificity. Together, we provided the first transcriptome comparison of medicinal leech species, signifying their molecular basis of muscle function and therapeutic efficiency, establishing a fundamental resource for future multi-omics integration, functional gene validation, and targeted molecular breeding to advance leech-derived medicinal applications.