Background: Advent of 2nd-generation TKIs has brought additional treatment options for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). To analyze the changes they have determined in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL mutation analyses done in our laboratory from 2004 through 2011. Methods: 781 tests on 258 pts were performed by direct sequencing. Results: 143 pts were analyzed because of imatinib resistance; 101 (71%) had one or more mutations (a single mutation in 91 pts; two mutations in 10 pts). Three mutation types were by far the most frequent: T315I (38 pts, 37%), E255K (19 pts, 18%) and Y253H (19 pts, 18%). Of 84 pts who had developed resistance to 2nd- or 3rd-line therapy with dasatinib, nilotinib or bosutinib after imatinib failure, 65 (77%) were positive for Bcr-Abl mutations; 30 (46%) carried multiple mutations (up to four) and in 19 of them (63%) this was consequence of multiple lines of therapy. The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib. Mutation analysis was also performed in 15 resistant pts enrolled in a study of dasatinib as 1st-line treatment of Ph+ ALL; 12 pts were positive, 11 of them had a T315I. Taking advantage of a next-generation sequencer (Roche 454), allowing a high sensitive and quantitative mutation scanning of Bcr-Abl, serially collected samples from 24 selected cases who developed mutations and resistance to one or more TKIs were retrospectively analyzed to study the kinetics of expansion of mutant clones. Results will be presented. Conclusions: Although 2^nd generation TKIs are more potent and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy. The high genetic instability fosters mutational events anytime during TKI treatment and some mutation types (T315I, Y253H) have been observed to emerge and take over very quickly (from <0.01% to 90% in one-two months). Supported by PRIN, AIL, AIRC, Fondazione CARISBO.