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BACKGROUND: A set of human remains unearthed near Ekaterinburg, Russia has been attributed to the Romanov Imperial Family of Russia and their physician and servants. That conclusion was officially accepted by the Russian government following publication of DNA tests that were widely publicized. The published study included no discussion of major forensic discrepancies and the information regarding the burial site and remains included irregularities. Furthermore, its conclusion of Romanov identity was based on molecular behaviour that indicates contamination rather than endogenous DNA. The published claim to have amplified by PCR a 1223 bp region of degraded DNA in a single segment for nine individuals and then to have obtained sequence of PCR products derived from that segment without cloning indicates that the Ekaterinburg samples were contaminated with non-degraded, high molecular weight, 'fresh' DNA. AIM: Noting major violations of standard forensic practices, factual inconsistencies, and molecular behaviours that invalidate the claimed identity, we attempted to replicate the findings of the original DNA study. SUBJECT: We analysed mtDNA extracted from a sample of the relic of Grand Duchess Elisabeth, sister of Empress Alexandra. RESULTS: Among clones of multiple PCR targets and products, we observed no complete mtDNA haplotype matching that reported for Alexandra. The consensus haplotype of Elisabeth differs from that reported for Alexandra at four sites. CONCLUSION: Considering molecular and forensic inconsistencies, the identity of the Ekaterinburg remains has not been established. Our mtDNA haplotype results for Elisabeth provide yet another line of conflicting evidence regarding the identity of the Ekaterinburg remains.