© Mary Ann Liebert, Inc. 2016. We examined baseline HIV-1 protease and reverse transcriptase sequences and HIV clinical parameters from 1,021 consecutive patients (814 male, 207 female) through the Royal Perth Hospital HIV service to investigate HIV-1 subtype diversity and local phylogenetic networks from 2000 to 2014. HIV-1 subtype B virus sequences were demonstrated in 619 (61%) of cases, with increasing non-B HIV-1 subtypes from 23.2% (2000-2003) to 48% (2008-2011) and 43% (2012-2014) (p <0.001), including the CRF01-AE subtype [6.6% (2000-2003) to 21.5% (2008-2011)] and HIV-1 C subtype [9.5% (2000-2003) to 20.2% (2008-2011)]. More HIV-1 B subtypes were assigned to phylogenetic clusters compared to non-B subtypes (34% vs. 18%; p <0.001), with larger clusters identified (cluster size >2: 135/211; 64% vs. 13/69; 19%; p = 0.001), including one cluster of 53 HIV-1 B subtype sequences that evolved from 2008 to 2014. Non-B subtype HIV-1 was associated with lower baseline CD4 T cell count (p = 0.005) but not plasma HIV-1 RNA levels (p = 0.31), suggesting relatively delayed diagnosis. Baseline viral load was strongly associated with calendar time [mean 18,620 copies/ml in 2000-2003; 75,858 copies/ml in 2012-2014 (p <0.001)], and was also associated with larger phylogenetic clusters (size >2) in adjusted analyses (p = 0.03). This study identifies a number of temporal trends over the past 15 years, including an increasing prevalence of non-B subtype HIV-1 that highlights the growing influence of migration and travel on the Australian HIV-1 epidemic and the associated increased role of heterosexual HIV-1 transmission in this context. At the same time, these data indicate that local transmission within predominantly male networks remains a challenging issue for HIV-1 prevention.