Piperaquine resistance in Plasmodium falciparum has emerged in Southeast Asia and is strongly associated with mutations in the pfcrt gene and amplification of pfplasmepsin2/3. This study assessed the frequency of pfcrt mutations and pfplasmepsin2/3 gene amplifications across Southeast Asia over 25 years, which is critical for tracking resistant parasites. A total of 898 P. falciparum isolates collected from Thailand, Myanmar, Cambodia, Laos, and Vietnam between 1995 and 2023 were analyzed for pfcrt mutations, pfplasmepsin2 copy number, and microsatellite variation around the pfcrt locus. During the study period, pfcrt mutations in Cambodia across the study period showed that H97Y had the highest prevalence at 15%, followed by T93S at 8%, while I218F was common in 24% of specimens in Srisaket and Ubon in Thailand at 24.44%, followed by G353V at 20%, T93S at 17%, F145I at 13%, and H97Y at 4%. In Tak, Thailand, a mutation was found only in 1995 with 40% at I218F and the remaining wild-type. Yala in Thailand and Kayin State in Myanmar remained wild-type. Vietnam showed frequent T93S at 21%. The prevalence of pfcrt mutations observed in this study changed throughout the study period. Over 65% of parasites with pfcrt mutations at positions 93, 97, 145, 218, 343, and 353 also had pfplasmepsin2 amplification. Temporal analysis revealed that in Cambodia, pfplasmepsin2 CNV emerged first, peaking at 88% prevalence in 2015 after the introduction of DHA-piperaquine, with the subsequent increase of pfcrt mutations H97Y and G353V. In Eastern Thailand, both pfcrt mutations and pfplasmepsin2 CNV were highly prevalent, exceeding 70% during 2015-2018. In Vietnam, rapid increases in both markers were observed after 2012, reaching peak by 2017. Microsatellite analysis revealed reduced genetic diversity around mutant pfcrt alleles, indicating selective sweeps. This study demonstrates that frequencies of pfcrt mutations and pfplasmepsin2 amplification linked to piperaquine resistance have changed over time, highlighting the importance of ongoing genetic monitoring to inform strategies for preserving artemisinin-based combination therapy efficacy.