Background <p>Among the crucial molecular markers contributing to multidrug resistance, the <i>Plasmodium falciparum multidrug resistance-1</i> gene (<i>Pfmdr1</i>) remains understudied as compared to other drug-resistant genes in terms of its genetic diversity and evolution pattern. This study presents a comprehensive analysis of <i>Pfmdr1</i> gene’s genetic diversity aiming to discern its dynamics, distribution and evolutionary trends especially in Indian and global populations.</p> Methods and Results <p>The <i>Pfmdr1</i> gene was amplified and sequenced from 256 <i>Plasmodium falciparum</i> mono-infected samples collected from 14 Indian states during the years 1993–2023. Analysis revealed six non-synonymous (N86Y, N86F, S137C, D144F, F157L and Y184F) and one synonymous mutation (G182G) in N-terminal fragment. Among these, N86F, S137C, D144F and F157L were novel findings. The most prevalent mutations were N86Y (18.91%), Y184F (64.71%) and G182G (GG<i>T</i> &gt; GG<i>G</i>) (59.24%; exclusive to India), with Y184F showing increasing trend when compared to N86Y over time. The mutation GG<i>T</i> &gt; GG<i>G</i> is experiencing a hitchhiking by Y184F mutation which is likely undergoing a selective sweep. High haplotype and nucleotide diversity were observed in most Indian states, particularly in Odisha and Delhi. However, a decrease in diversity was noted in samples from 2020 onwards throughout India. Globally <i>Pfmdr1</i> showed tendency of negative selection, except for populations from Liberia, Nigeria, Sudan and Central African Republic. Notably, samples from Sudan depicted a distinct haplotype and population structure compared to other countries.</p> Conclusions <p>These findings contribute significantly to our understanding of the genetic structure and evolutionary trends of <i>Pfmdr1</i>, which can help to strengthen the current malaria control policies for emergence of drug resistance.</p>