(1) Background: This study was aimed at identifying the Colletotrichum species associated with twig and shoot dieback of citrus, a new syndrome occurring in the Mediterranean region and also reported as emerging in California. (2) Methods: Overall, 119 Colletotrichum isolates were characterized. They were recovered from symptomatic trees of sweet orange, mandarin and mandarin-like fruits during a survey of citrus groves in Albania and Sicily (southern Italy). (3) Results: The isolates were grouped into two distinct morphotypes. The grouping of isolates was supported by phylogenetic sequence analysis of two genetic markers, the internal transcribed spacer regions of rDNA (ITS) and β-tubulin (TUB2). The groups were identified as Colletotrichum gloeosporioides and C. karstii, respectively. The former accounted for more than 91% of isolates, while the latter was retrieved only occasionally in Sicily. Both species induced symptoms on artificially wound inoculated twigs. C. gloeosporioides was more aggressive than of C. karstii. Winds and prolonged drought were the factor predisposing to Colletotrichum twig and shoot dieback. (4) Conclusions: This is the first report of C. gloeosporioides and C. karstii as causal agents of twig and shoot dieback disease in the Mediterranean region and the first report of C. gloeosporioides as a citrus pathogen in Albania.