The patterns of transmitted drug-resistant (TDR) HIV-1 variants, non-B subtype spread, and epidemiological trends were evaluated either in seroconverters or in newly diagnosed individuals in Italy over a 13-year period. We analyzed 119 seroconverters, enrolled from 1992 to 2003 for the CASCADE study, and 271 newly diagnosed individuals of the SPREAD study (2002-2005), of whom 42 had a known seroconversion date. Overall, TDR was 15.1% in the CASCADE and 12.2% in the SPREAD study. In the 1992-2003 period, men having sex with men (MSMs) and heterosexuals (HEs) were 48.7% and 36.8%, respectively; TDR was found to be higher in MSMs compared to HEs (78.9% vs. 21%, p = 0.006). The same groups were 39.1% and 53.3% in the SPREAD study; however, no association was detected between modality of infection and TDR. Overall, 9.2% and 22.1% of individuals harbored a non-B clade virus in the CASCADE and SPREAD study, respectively. As evidence of onward transmission, 40% (24/60) of non-B variants were carried by European individuals in the latter study; among these patients the F1 subtype was highly prevalent (p = 0.00001). One of every eight patients who received a diagnosis of HIV-1 in recent years harbored a resistant variant, reinforcing the arguments for baseline resistance testing to customize first-line therapy in newly infected individuals. The spread of non-B clades may act as a dilution factor of TDR concealing the proportion of TDR in seroconverters and MSMs.