Pooley powered through the 22.9 km course in the port city of Geelong in 32 minutes 48.44 seconds to finish 15.7 seconds faster than German silver medallist Judith Arndt. Linda Villumsen of New Zealand took bronze.
Pooley, who took silver in the time trial at the 2008 Beijing Olympics, had to overcome a last-minute hitch when race officials found a discrepancy with her bike just before the starting line and ordered her to change it.
French cycling great Jeannie Longo-Ciprelli defied her 51 years with an extraordinary ride to finish fifth, ahead of a field of riders half her age.
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Pooley will bid for the double when she races in the women's road race on Saturday.
