Fragmented and badly damaged commingled human remains present a tough challenge for their identification pursuits in forensic anthropology. Thousands of unknown human remains along with items of contextual identity, reportedly belonging to 282 Indian sepoys killed in 1857, were exhumed mon-scientifically from a disused ancient well at Ajnala (Amritsar, India). In this manuscript, the non-scientific excavation of unknown human remains from the abandoned well, their forensic anthropological strategies for identification purposes, challenges being faced and future possibilities of their biological profiling have been discussed. Multiple methods and techniques like anthropological examinations, odontological profiling, radiological analyses, stable isotope and mitochondrial DNA (mtDNA) analyses were applied to few bones and teeth collected from the Ajnala skeletal assemblage. Though majority of studied bones and teeth were found belonging to adult males, very few of them had morphological, osteological and molecular features questioning the authenticity and validity of the written records. Due to certain ambiguous findings or gaps observed between the anthropological analyses of the Ajnala skeletal remains and the reported versions about their affiliations; certain advanced radiological, chemical and molecular techniques were applied to estimate their probable age, sex and populational affinity. The obtained radiological, isotopic and molecular signatures of the remains were compared with the available databases to estimate their affinity with the individuals of geographic area to whom the remains reportedly belonged to. The maternally inherited mtDNA haplogroup assignments, and stable isotope analysis of carbon and oxygen suggested that the studied human remains belonged to the individuals from West Bengal, Bihar, Odisha, Awadh (presently in Uttar Pradesh) and parts of Meghalaya and Manipur as potential regions of their geographic identity and thus, attributing the victims to be non-local to the site. However, merely on the basis of forensic anthropological examinations of very few bones and teeth (collected out of a huge collection of thousands of bones and teeth); it would be just an unqualified and sweeping conclusion to claim their identity as adult or non-adult, male or female, local or non-local, victims of 1857 mass killings or to the victims of ceremonial sacrifices or criminal activities committed in the past. A sufficient number of bones and teeth along with items of personal identity needs to be examined with multiple scientific techniques to arrive at some valid conclusions about their biological identity.