Ropy defect of pasteurized fluid milk is a type of spoilage which manifests itself by an increased viscosity, slimy body, and string-like flow during pouring. This defect has, among other causes, been attributed to the growth, proliferation and exopolysaccharide production by coliform bacteria, which are most commonly introduced in milk as post-pasteurization contaminants. As we identified both Klebsiella pneumoniae subsp. pneumoniae and Rahnella inusitata that were linked to a ropy defect, the goal of this study was to characterize three K. pneumoniae subsp. pneumoniae strains and two R. inusitata for (i) their ability to grow and cause ropy defect in milk at 6 and 21°C and to (ii) probe the genetic basis for observed ropy phenotype. While all K. pneumoniae subsp. pneumoniae and R. inusitata strains showed net growth of >4 log10 over 48 h in UHT milk at 21°C, only R. inusitata strains displayed growth during 28-day incubation period at 6°C (>6 log10). Two out of three K. pneumoniae subsp. pneumoniae strains were capable of causing the ropy defect in milk at 21°C, as supported by an increase in the viscosity of milk and string-like flow during pouring; these two strains were originally isolated from raw milk. Only one R. inusitata strains was able to cause the ropy defect in milk; this strain was able to cause the defect at both 6 and 21°C, and was originally isolated from a pasteurized milk. These findings suggest that the potential of K. pneumoniae subsp. pneumoniae and R. inusitata to cause ropy defect in milk is a strain-dependent characteristic. Comparative genomics provided no definitive answer on genetic basis for the ropy phenotype. However, for K. pneumoniae subsp. pneumoniae, genes rffG, rffH, rfbD, and rfbC involved in biosynthesis and secretion of enterobacterial common antigen (ECA) could only be found in the two strains that produced ropy defect, and for R. inusitata a set of two glycosyltransferase and flippase genes involved in nucleotide sugar biosynthesis and export could only be identified in the ropy strain. While these results provide some initial information for potential markers for strains that can cause ropy milk, the relationship between genetic content and ropiness in milk remains poorly understood and merits further investigation.