A novel mutation, insertion/deletion (InDel) was found in the porcine prolactin gene exon 5 coding for the 3’UTR. Polymorphism was detected and identified using PCR-MSSCP and DNA sequencing techniques, and then analysed with PCR-RFLP using HpyCH4III endonuclease. Inserted/deleted was an 11-bp TGTTTCTTAAC motive that occurred once or twice at position 201-222 in the amplified DNA fragment. The investigations covered 150 gilts of the Puławska breed from the herds under the Preserve Breeding Project. The frequencies of the inserted and deleted PRL gene variants were 0.34 and 0.66, respectively. The estimated value of heterozygosity (Ĥ) was 0.253 and the polymorphism information content (PIC) amounted to 0.348.