During a series of sampling in 2008 and 2009, stem rot disease was detected in Hylocereus polyrhizus plantations in Malaysia, with symptom appeared as circular, brown sunken lesion with orange sporodochia and white mycelium formation on the lesion surface. Eighty-three isolates of Fusarium were isolated from 20 plantations and were morphologically identified as F. proliferatum based on the variability of colony appearance, pigmentation, growth rate, length of chains, production of bluish sclerotia, concentric ring aerial mycelium and sporodochia. Three species-specific primers, namely ITS1/proITS-R, PRO1/2 and Fp3-F/4-R successfully produced PCR products and confirmed that the isolates from stem rot of H. polyrhizus were F. proliferatum isolates. From BLAST search of translation elongation factor 1-alpha (TEF1-α) sequences, the isolates showed 99–100% similarity with F. proliferatum deposited in GenBank which further confirmed that the isolates were F. proliferatum. The results from amplification of MAT-allele specific primers indicated that 14.5% of F. proliferatum isolates carried MAT-1 allele and 85.5% carried MAT-2. Crossing results showed that all 83 F. proliferatum isolates were male fertile showing positive crosses with the tester strains of MATD-1 and MATD-2. Perithecia oozing ascospore were produced. Forty isolates as representative were evaluated for pathogenicity test, produced rot symptoms similar to those observed in the fields which confirmed the isolates as the causal agent of stem rot of H. polyrhizus. To our knowledge, this is the first report of stem rot of H. polyrhizus caused by F. proliferatum in Malaysia.