Objective To identify the frequencies (F) of ferredoxin and nitroreductase mutations on Iranian clinical isolates of Giardia lamblia in order to predict whether the nitazoxanide can be prescribed as suitable drug for symptomatic to metronidazole-resistant giardiasis. Methods Forty Giardia lamblia isolates as of 38 symptomatic and two metronidazole-resistant patients were collected from Iran. DNAs were extracted and amplified by targeting ferredoxin and GlNR genes. The amplicons were directly sequenced to determine gene mutations. Results The various amino acid substitutions (F: 20%, Haplotype diversity: 0.891, Tajima's D: −0.440 13) were identified by analyzing ferredoxin gene in four symptomatic and two resistant isolates. Only two haplotypes (F: 5%, HD: 0.345; Tajima's D: 0.778 15) characterized in metronidazole-resistant isolates of GlNR, however, no point mutations was found in symptomatic isolates. Conclusions Non-synonymous mutations of ferredoxin oxidoreductase gene reduce translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression and its activity. This leads to decrease in metronidazole drug delivery into the cells. Mutations in these isolates may lead to their resistance to metronidazole. No to low synonymous mutations of GlNR demonstrates that nitazoxanide can be prescribed as promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in Iranian clinical isolates.