Rust fungi are widespread plant pathogens that infect a broad range of species, including major crops. One of the most significant threats to wheat production is Puccinia striiformis f.sp. tritici (Pst), a fungal pathogen specialized in infecting wheat causing the disease stripe rust. Over the past few decades, four Pst lineages have been introduced to Australia. Relative frequency of these lineages and pathotypes causing disease changed over time with more recently introduced lineages replacing older introductions. Like many other rust fungi, Pst is dikaryotic and maintains distinct copies of its genome within two separate nuclei, a unique genetic organization that may contribute to its adaptability and virulence. To better understand the genomic basis of adaptation, we generated high-quality, haplotype-phased genome assemblies for all four major Australian lineages and from isolates from a long-term asexual lineage in Europe using PacBio HiFi and Oxford Nanopore long-read sequencing. By integrating genomic data with long-read transcriptome sequencing, epigenetic modifications and virulence profiles, we identified candidate effector genes that may interact with specific Yr resistance genes in wheat. To test these interactions, we applied our newly developed wheat protoplast assay, expressing candidate effectors in protoplasts from different wheat cultivars. Our findings not only highlight key genes involved in host-pathogen interactions but also shed light on the genome biology of Pst. Understanding how its dikaryotic state influences disease severity and rapid adaptation to variable and changing agricultural environments will provide important insights for developing more durable disease resistance strategies in wheat.