The alternative oxidase (AOX) is a key enzyme of the alternative respiration pathway and is involved in plant response to a variety of environmental stresses. The genes encoding for this enzyme are highly polymorphic and thus likely good candidates for molecular marker development to assist plant breeding on stress tolerance. In carrot, DcAOX1 gene has shown to be the most responsive to environmental changes. Therefore, we have studied its allelic variation in different carrot materials, including commercial cultivars, inbred lines, carrot subspecies and carrot crop wild relatives. This work is thus a first step to develop molecular markers based on AOX1 gene. Sequence comparison revealed the existence of high number of single nucleotide polymorphisms (SNPs), as well as several insertion/deletion events (InDels), especially in exon 1 and intron 1. Intron 1 showed to be the most polymorphic region and harbored an insertion event of 400 bp, which had highly divergent sequences depending on the carrot genotype. The insertion was located in a region of single tandem repeats (SSR) that was also polymorphic between genotypes. This intronic insertion, together with its associated SSRs, arises as a good source of candidate functional markers (FMs) that, once validated, could greatly contribute to marker assisted selection of new carrot genotypes.