Abstract: This study analysed mitochondrial phosphoenol-pyruate carboxykinase (PEPCK-M) gene as a candidate QTL for egg production traits in chickens. Single Strand Conformational Polymorphism (SSCP) of a 300 bp DNA fragment, from exon 9 of samples from an egg laying North American commercial White Leghorn stock, revealed a total of 6 different single strand conformers, indicative of 3 alleles. Subsequent DNA sequencing found a total of 4 base changes in this fragment between these alleles (called A1, A2 and A3) when compared to the reference sequence published online. The A1 allele had one transition mutation of T to C at position 1700. The A2 allele had accumulated three transition mutations: T to C at position 1578, A to G at position 1647 and T to C at position 1650. Transition mutation of T to C at position 1578 of the A2 allele results in the loss of an AccI site, hence, producing a de novo RFLP. Analysis of 358 female individuals from this strain showed that the population is highly polymorphic at this site. The effect of PEPCK-M genotypes at this site, namely AccI -/-, AccI +/- and AccI +/+, was tested on three traits, age at first egg, egg production rate and egg number. Least square analysis showed that exon 9 RFLP significantly affects age at first egg (p<0.05). Egg production rate and egg number traits were not affected by different genotypes at this position. The data also indicates an over-dominance effect for the associated trait.