Lymphogranuloma venereum (LGV) is a sexually transmitted infection caused by Chlamydia trachomatis serovars L1, L2 and L3. Consequently, more specific and sensitive detection methods that are rapid and inexpensive are necessary to differentiate between C. trachomatis serovars. The purpose of this study was to identify and differentiate LGV-related C. trachomatis serovars from rectal swabs using high-resolution melting analysis (HRMA) and multiplex allele-specific polymerase chain reaction (MAS-PCR). Fifteen clinical samples from patients in Sydney were first screened and confirmed as C. trachomatis by using the COBAS AMPLICOR PCR analyser. The same samples were assayed for C. trachomatis and LGV by HRMA and MAS-PCR of the polymorphic membrane protein H (pmpH) gene. Both methods indicated that two of 15 samples were serovar L2 and the remainder (13/15) other C. trachomatis serovars. Both HRMA and MAS-PCR are inexpensive, rapid, easy methods that are useful tools for the identification of LGV in clinical and research laboratories.