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Abstract: Sampling performed in flower buds of citrus in Colombia allowed the detection of the presence of Prodiplosis longifila Gagné and Prodiplosis floricola Felt (Diptera: Cecidomyiidae). The polyphagous P. longifila is a pest of Solanaceae, but whether both species cause economic losses in citrus is unknown. These congeneric species are indistinguishable in the larval stage, and their morphological identification, which is complex, is performed on adult males. This study comprises a morphological description and characterization of the damage generated by P. floricola larvae feeding on the flowers of key lime, Citrus × aurantiifolia (Christm.) Swingle (pro. sp.), and Tahiti lime, Citrus × latifolia Tanaka ex Q. Jiménez (Rutaceae), as well as a population genetical analysis of the insect. Prodiplosis floricola was found in the localities of Támesis (Antioquia), Garzón (Huila), and Palmira and Zarzal (Valle del Cauca). These populations were characterized by COI and ITS2 molecular markers, with a moderate genetic structure found that cannot be explained by the geographic distance between the populations. The haplotype distribution pattern indicates that the populations are composed of highly differentiated haplotypes of equivalent frequencies, which suggests that the Colombian populations of P. floricola are products of multiple introductions and that their dispersion between populations is attributable to anthropic transport. Citrus in Colombia constitutes a new host for P. floricola and increases the known geographical range of the insect, as it has only been previously reported in Brazil and the USA. The molecular markers used in the present study are useful for early diagnosis of P. floricola in the larval stage and for future research on the population dynamics in citrus. [ABSTRACT FROM AUTHOR]