Xylella fastidiosa subsp. pauca is genetically diverse and has many vector species. However, there is limited information on vector specificity and efficiency for different sequence types (STs) within the pathogen subspecies. Both STs of X. fastidiosa and vectors differ in their associations with plants; therefore, assessment of vector competence should include the standardized vector acquisition ability of bacteria from artificial diets. This work aimed to adapt and validate an in vitro acquisition system for strains of X. fastidiosa that cause citrus variegated chlorosis, and to compare the transmission efficiency of STs of subsp. pauca by different species of sharpshooter vector. First, acquisition and transmission of ST13 by Bucephalogonia xanthophis and Macugonalia leucomelas was tested using an artificial diet with bacteria grown on minimum defined medium (X. fastidiosa medium) with or without 1% galacturonic acid (GA). Subsequently, four sharpshooter species (B. xanthophis, M. leucomelas, M. cavifrons, and Sibovia sagata) were compared as vectors of ST13 acquired from artificial diets, and four STs of subsp. pauca (11, 13, 65, and 70) were tested for acquisition and transmission by M. leucomelas. The artificial system allowed efficient acquisition and transmission of ST13 to plants, with no differences between the media tested. ST13 was transmitted more efficiently by B. xanthophis and M. leucomelas when compared with M. cavifrons and S. sagata. Different STs influenced acquisition and transmission rates by M. leucomelas. The differences in vector competence, despite the standardized acquisition system, suggest that ST–vector foregut or vector–plant interactions may influence bacterial acquisition, retention and inoculation by the insect.