ABSTRACT This study reports the first confirmed case of Myxobolus koi parasitic infection in koi ( Cyprinus carpio koi ) imported into Thailand. Infected fish exhibit clinical signs during the quarantine process, including lethargy, tachypnea, flared gill opercula and excessive mucus production in the gills. The gross pathological findings included swollen and pale gills interspersed with white nodules. Microscopic examination of gill biopsies revealed mucus clumps and numerous pyriform myxospores, with no evidence of other parasites. Molecular analysis confirmed the presence of Myxobolus spp. DNA through PCR amplification targeting the 18S SSU rRNA. Phylogenetic analysis of 776 bp sequences from our representative isolates (CU01, CU02 and CU03) revealed 100% nucleotide identity with M. koi sequences from goldfish in China (OM757920) and koi in the USA (FJ841887), which formed a distinct clade with strong bootstrap support. Haematological parameters were compared between infected and healthy fish from the same batch, revealing significantly elevated levels of creatine and ALT ( p < 0.05) in the infected group. Histopathological examination revealed severe gill damage, with plasmodia infiltrating and disrupting the lamellar architecture. The well‐delineated pink fibrous septa encapsulated mature myxospores and pansporoblasts. Scanning and transmission electron microscopy revealed that the M. koi spores, measuring 5.2 × 2.92 μm, were oval, elongated and pyriform, with coiled polar filaments enclosed within two polar capsules. These findings document the occurrence of M. koi in Thailand, underscore its transboundary transmission via international trade and highlight the need for enhanced biosecurity measures to mitigate the spread of aquatic pathogens.