The skeletal remains were found by a bushwalker on a remote property in Gooloogong in central western New South Wales state in May 2009.
Testing confirmed in April this year that the bones were those of Judith Bartlett, who was last seen 145 kilometres away in Bathurst in 1964. They are believed to have been there since the 1960s.
"We've now commenced a homicide investigation ... Given the nature of the inquiries we've made," Detective Inspector Denise Godden told reporters.
Police said her identification brought some peace to the family, who had never known whether their mother had simply left them or been the victim of an accident or foul play.
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"Our mother loved us," daughter Frances Ryan said through tears today.
"My brothers and I have found out that our mother didn't leave us. She didn't stop loving us. She was taken by a thief in the night."
Bartlett was last seen heading to work at The Royal Hotel at Bathurst in March 1964.
"We are hoping that we can find a large number of answers for the family. Whether or not we get to charge someone after all this time ... Is a hard question to answer but someone out there knows something.