The myotendinous junction (MTJ) is a specialised interface for transmitting high forces between muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses and the tissue was separated through a sequential cryo-sectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-SNE plots were used to select the muscle, MTJ and tendon samples from 5 horses for RNA-sequencing. The expression of previously known and unknown genes identified through RNA-sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA-sequencing identified expression of a panel of 61 genes enriched at the MTJ. 48 of these genes were novel for the MTJ, and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known (COL22A1, NCAM, POSTN, NES, OSTN) and previously undescribed (MNS1 and LCT) MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.