The exotic tephritid Bactrocera invadens (syn. Bactrocera dorsalis) and the native Ceratitis species constitute serious pests and major impediment to Citrus production in Nigeria. Lack of accurate and reliable diagnostic methods independent of the life stages intercepted for both genera hampers implementation of appropriate early management programmes to avoid economic losses and delays in quarantine decision regarding these pests. We used a PCR amplification technique to successfully establish RFLP patterns of the COI coding gene to differentiate the exotic B. invadens from the native Ceratitis species infesting sweet oranges in Nigeria. The universal barcoding primer pair LCO1490/HCO2198 was used to amplify 658 bp-long fragment of the mitochondrial COI coding gene. The amplified fragment was analysed by automated direct sequencing and RFLP. Intraspecific variation in nucleotide sequences of B. invadens was very low with not more than 2 bp substitutions in four out of 31 individuals sequenced. Among the Ceratitis species intraspecific variation was also very low with at most 4 bp substitutions in few of the 32 individuals sequenced. The restriction enzymes Rsa I and Hsp92 II produced patterns that clearly and unambiguously separated B. invadens from Ceratitis spp. DNA barcoding was also used to confirm the identities of the tephritid species analysed. The molecular method established in this study will enhance easy monitoring, early detection of species involved, and implementation of appropriate management programmes that effectively reduce yield loses in Citrus production in Nigeria.