Feline caliciviruses (FCVs) are potential etiologic agents in feline idiopathic lower urinary tract disease (I-LUTD). By means of a modified virus isolation method, we examined urine obtained from 28 male and female cats with nonobstructive I-LUTD, 12 male cats with obstructive I-LUTD, and 18 clinically healthy male and female cats. All cats had been routinely vaccinated for FCV. Two FCVs were isolated; 1 (FCV-U1) from a female cat with nonobstructive I-LUTD, and another (FCV-U2) from a male cat with obstructive I-LUTD. To determine the genetic relationship of FCV-U1 and FCV-U2 to other FCVs, capsid protein gene RNA was reverse transcribed into cDNA, amplified, and sequenced. Multiple amino acid sequence alignments and phylogenetic trees were constructed for the entire capsid protein, hypervariable region E, and the more conserved (nonhypervariable) regions A, B, D, and F. When compared to 23 other FCV isolates with known biotypes, the overall amino acid sequence identity of the capsid protein of FCV-U1 and FCV-U2 ranged from 83 to 96%; identity of hypervariable regions C and E ranged from 58 to 85%. Phylogenetically, FCV-U1 clearly separated from other FCV strains in phenograms based on nonhypervariable regions. In contrast, FCV-U2 consistently segregated with the Urbana strain in all phenograms. Clustering of isolates by geographic origin was most apparent in phenograms based on nonhypervariable regions. No clustering of isolates by biotype was apparent in any phenograms. Our results indicate that FCV-U1 and FCV-U2 are genetically distinct from other known vaccine and field strains of FCV.