This opinion addresses a request from the European Commission to evaluate whether heterogeneous populations of Xylella fastidiosa subsp. pauca have been found in Apulia (Italy) in addition to the strain named CoDiRO. After reviewing the most recent scientific literature and conducting further sequence analysis of the housekeeping genes used to genotype X. fastidiosa from Apulia, the EFSA Panel on Plant Health concluded that the currently available scientific evidence does not support the notion of the existence of heterogeneous populations of X. fastidiosa in Apulia. To reach this conclusion, several lines of arguments have been considered: (i) the currently accepted multilocus sequence analysis (MLST) approach provides a robust and sensitive framework to estimate X. fastidiosa diversity, which may be further improved by whole genome sequence analysis; (ii) all scientific papers evaluated on X. fastidiosa in Apulia come to the conclusion that - notwithstanding host and location - X. fastidiosa in Apulia is ST53; and (iii) a single article by Elbaino et al. (2014) provides contradictory statements. After assessment of the methodology and re-analysis of the sequence information underlying the conflicting statement, the Panel considered that the data presented in that article do not support firm conclusions on the existing diversity of isolates. The Panel further considered that MLST data are currently available for only 18 Apulian isolates, and thus more data are needed to study further X. fastidiosa diversity, strain evolution and route(s) of introduction. From all evidence currently available, the Panel concludes that X. fastidiosa isolates involved in the current epidemic in Apulia belong to a single sequence type, ST53. There is no other information at this moment supporting the notion that, in addition to the strain named CoDiRO, other diversity exists.