Coenzyme Q10 (CoQ10) is an industrially important molecule having nutraceutical and cosmeceutical applications. CoQ10 is mainly produced by microbial fermentation and the process demands the use of strains with high productivity and yields of CoQ10. During strain improvement program consisting of sequential induced mutagenesis, rational selection and screening process, a mutant strain UF16 was generated from Sporidiobolus johnsonii ATCC 20490 with 2.3-fold improvements in CoQ10 content. EMS and UV rays were used as mutagenic agents for generating UF16 and it was rationally selected based on atorvastatin resistance as well as survival at free radicals exposure. We investigated the genotypic and phenotypic changes in UF16 in order to differentiate it from wild type strain. Morphologically it was distinct due to reduced pigmentation of colony, reduced cell size and significant reduction in mycelial growth forms with abundance of yeast forms. At molecular level, UF16 was differentiated based on PCR fingerprinting method of RAPD as well as large and small-subunit rRNA gene sequences. Rapid molecular technique of RAPD analysis using six primers showed 34 % polymorphic fragments with mean genetic distance of 0.235. The partial sequences of rRNA-gene revealed few mutation sites on nucleotide base pairs. However, the mutations detected on rRNA gene of UF16 were less than 1 % of total base pairs and its sequence showed 99 % homology with the wild type strain. These mutations in UF16 could not be linked to phenotypic or genotypic changes on CoQ10 biosynthetic pathway that resulted in improved yield. Hence, investigating the mutations responsible for deregulation of CoQ10 pathway is essential to understand the cause of overproduction in UF16. Phylogenetic analysis based on RAPD bands and rRNA gene sequences coupled with morphological variations, exhibited the novelty of mutant UF16 having potential for improved CoQ10 production. © 2014 Association of Microbiologists of India.