It is well documented that prolactin (PRL) plays important roles in controlling vertebrate reproduction, growth and development, metabolism, osmo-regulation, immunomodulation, and behavior. PRL is synthesized mainly by pituitary lactotrophs and regulates these physiological processes through an endocrine route in vertebrates; however, there has been increasing evidence that in mammals, PRL is also expressed in the extra-pituitary tissues such as brain, immune cells, skin and mammary gland, and plays important autocrine/paracrine roles in these tissues (Ben-Jonathan et al. 1996, Harvey et al. 2012). In contrast, evidence for the extra-pituitary expression of PRL in non-mammalian vertebrates was limited. Here, we summarize our recent progress on the expression of PRL gene (PRL) and a novel PRL-like gene (PRL-L) in the extra-pituitary tissues of chickens (Gallus gallus) and zebrafish (Danio rerio). Our data shows that: 1) cPRL mRNA is expressed in several extra-pituitary tissues of chickens, including testes and spinal cord (detected by quantitative real-time RT-PCR), and its expression in the testes is stage-dependent with relatively high mRNA levels noted at the post-hatch stage, whereas cPRL mRNA seems to be highly expressed in the spinal cord at all stages examined (from embryonic day 14 to adult stage); 2) cPRL mRNA is also expressed in the skin of chicken embryos or post-hatching chicks, and its expression is likely associated with feather development; 3) a novel prolactin-like protein (cPRL-L) encoded by PRL-L gene (EU327778) is present in chickens. This putative PRL-L shares 30-35% amino acid sequence identity with PRL of chickens, rats and humans. Functional study further proved that recombinant cPRL-L protein produced in E. coli can activate chicken PRL receptor (cPRLR) expressed in human hepatocellular carcinoma (HepG2) cells, indicating that cPRL-L functions as an additional ligand for cPRLR; 4) Unlike cPRL, cPRL-L is widely expressed in adult chicken extra-pituitary tissues with only minimal expression detected in the pituitary gland by RT-PCR. In day-7 chicken embryos, the mRNA expression of cPRL-L, but not cPRL, was also detected in all extra-pituitary tissues examined; 5) Congruent to our findings in chickens, a functional prolactin-like protein (zfPRL-L, EU327779) was also identified in zebrafish, and RT-PCR assay revealed that zfPRL-L mRNA is expressed in the extra-pituitary tissues including brain, gill, muscle, ovaries and testes. Taken together, the wide expression of PRL and/or PRL-L in the extra-pituitary tissues of chickens (or zebrafish) suggests their potential paracrine/autocrine roles in these tissues of non-mammalian vertebrates.