The marine polychaete Chaetopterus variopedatus (Renier) (family Chaetopteridae) is a cosmopolitan species complex, consisting of distinct populations/ subspecies. The worms release glowing (460 nm) clouds of mucus when disturbed, and their parapodia often glow brightly. Currently, it is still unclear how exactly the bioluminescence system of these polychaetes functions. It has been previously assumed that the C. variopedatus luciferase may be used for detection of ferroptosis, the recently explored pathway of programmed cell death, resulting from accumulation of the ferrous ions. This study was aimed to extract and characterize the C. variopedatus luciferases, as well as to compare luciferases obtained from C. variopedatus of different populations. When extracting the enzyme responsible for bioluminescence from the frozen samples of Brazilian C. variopedatus using the improved method, two active luciferases, L1 and L2, were obtained. We assumed that one of the listed above luciferases was responsible for luminescence of the mucus and the other luciferase was responsible for luminescence in parapodia, and used the method for the distinct samples of mucus and parapodia of the living Far Eastern C. variopedatus. However, mucus of the latter turned out to be non-glowing. It is shown that luciferase L2 is responsible for luminescence in the parapodia of the C. variopedatus polychaete, since this luciferase has been found in the total biomass of Brazilian polychaetes and parapodia of Far Eastern polychaetes. Luminescence of the Brazilian C. variopedatus mucus is attributed to the functioning of luciferase L1, which is lacking in the mucus of the Far Eastern subspecies. The range of luciferase isoforms in polychaetes C. variopedatus depends on the place of origin.