19 DNA barcoding is highly effective for identifying specimens once a reference sequence library is 20 available for the species assemblage targeted for analysis. Despite the great need for an improved 21 capacity to identify the insect pests of crops, the use of DNA barcoding is constrained by the lack 22 of a well-parameterized reference library. The current study begins to address this limitation by 23 developing a DNA barcode reference library for the pest aphids of Pakistan. It also examines the 24 affinities of these taxa with conspecific taxa from other geographic regions based on both 25 conventional taxonomy and Barcode Index Numbers (BINs). A total of 809 aphids were 26 collected from 123 plant species at 87 sites across Pakistan. Morphological study and DNA 27 barcoding allowed 774 specimens to be identified to one of 42 species while the others were 28 placed to a genus or subfamily. The 801 sequences obtained from these specimens were assigned 29 to 52 BINs whose monophyly were supported by neighbor-joining (NJ) clustering and Bayesian 30 inference. The 42 species were assigned to 41 BINs with 38 showing BIN concordance; one 31 species (Rhopalosiphum padi) was assigned to two BINs, while two others (Aphis affinis, Aphis 32 gossypii) were assigned to the same BIN, while one species (Aphis astragalina) lacked a 33 qualifying sequence. The 42 Linnaean species were represented on BOLD by 7,870 records from 34 69 countries. Combining these records with those from Pakistan produced to 60 BINs with 12 35 species showing a BIN split and three a BIN merger. Geo-distance correlations showed that 36 intraspecific divergence values for 18 of 37 species were not affected by the distance between 3 37 populations. Forty four of the 52 BINs from Pakistan had counterparts in 73 countries across six 38 continents, documenting the broad distributions of pest aphids. 39 Key words: Hemiptera, plant-pest, DNA barcoding, Barcode Index Number, COI, Pakistan 4 40 Introduction 41 Although aphids (Hemiptera: Aphididae) are important plant pests, their life stage diversity and 42 phenotypic plasticity have constrained the development of effective taxonomic keys [1,2]. With 43 over 4,700 described species, the Aphididae is the largest family within the Aphidoidea [3]. Most 44 pest aphids belong to the subtribe Aphidina which includes 670 described species [3,4]. Nearly 45 100 aphid species have been listed as serious agricultural pests; they attack more than 300 plant 46 species [5,6], and lower crop yield by direct feeding and by transmitting viral diseases [7].47 Sibling species complexes are common in many pest aphids [8]. Very often, these species are 48 morphologically identical but genetically distinct [9]. They often include anholocyclic biotypes 49 (=clones) with differing host preferences and varying competency for disease transmission 50 [10,11]. Species identification is so challenging that taxonomic keys are either ineffective or only 51 useful for a particular geographic area or taxonomic group 12]. These deficits h...