Agglutinin like sequence (Als) cell-wall proteins play a key role in adhesion and virulence of Candida species. Compared to the well-characterized Candida albicans ALS genes, little is known about ALS genes in the Candida parapsilosis species complex. Three incomplete ALS genes were identified in the genome sequence for Candida orthopsilosis strain 90-125 (GenBank assembly ASM31587v1): CORT0C04210 (named CoALS4210), CORT0C04220 (CoALS4220) and CORT0B00800 (CoALS800). To complete the gene sequences, new data were derived from strain 90-125 using Illumina (short-read) and Oxford Nanopore (long-read) methods. Long-read sequencing analysis confirmed the presence of 3 ALS genes in C. orthopsilosis 90-125 and resolved the gaps located in repetitive regions of CoALS800 and CoALS4220. In the new genome assembly (GenBank PQBP00000000), the CoALS4210 sequence was slightly longer than in the original assembly. C. orthopsilosis Als proteins encoded features well-known in C. albicans Als proteins such as a secretory signal peptide, N-terminal domain with a peptide-binding cavity, amyloid-forming region, repeated sequences, and a C-terminal site for glycosylphosphatidylinositol anchor addition that, in yeast, suggest localization of the proteins in the cell wall. CoAls4210 and CoAls800 lacked the classic C. albicans Als tandem repeats, instead featuring short, imperfect repeats with consensus motifs such as SSSEPP and GSGN. Quantitative RT-PCR showed differential regulation of CoALS genes by growth stage in six genetically diverse C. orthopsilosis clinical isolates, which also exhibited length variation in the ALS alleles, and strain-specific gene expression patterns. Overall, long-read DNA sequencing methodology was instrumental in generating an accurate assembly of CoALS genes, thus revealing their unconventional features and first insights into their allelic variability within C. orthopsilosis clinical isolates.